Methods and products for tissue repair

ABSTRACT

Methods and devices for the repair of articular tissue using collagen material are provided. Compositions of collagen material and related kits are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application is a continuation application which claims the benefitunder 35 U.S.C. §120 of U.S. application Ser. No. 12/412,692, entitled“METHODS AND COLLAGEN PRODUCTS FOR TISSUE REPAIR” filed on Mar. 27,2009, which is herein incorporated by reference in its entirety.Application Ser. No. 12/412,692 is a continuation of InternationalPatent Application Serial No. PCT/US2007/021009, entitled “METHODS ANDCOLLAGEN PRODUCTS FOR TISSUE REPAIR” filed Sep. 28, 2007, which isherein incorporated by reference in its entirety. ApplicationPCT/US2007/021009 claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 60/847,743, entitled “METHODS ANDPRODUCTS FOR TISSUE REPAIR” filed on Sep. 28, 2006, which is hereinincorporated by reference in its entirety.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under NIH K02 AR049346.Accordingly, the Government may have certain rights in this invention.

FIELD OF THE INVENTION

The invention relates generally to methods and devices for the repair ofarticular tissue using collagen materials.

BACKGROUND OF THE INVENTION

Intra-articular tissues, such as the anterior cruciate ligament (ACL),do not heal after rupture. In addition, the meniscus and the articularcartilage in human joints also often fail to heal after an injury.Tissues found outside of joints heal by forming a fibrin clot, whichconnects the ruptured tissue ends and is subsequently remodeled to formscar, which heals the tissue. Inside a synovial joint, a fibrin cloteither fails to form or is quickly lysed after injury to the knee, thuspreventing joint arthrosis and stiffness after minor injury. Jointscontain synovial fluid which, as part of normal joint activity,naturally prevent clot formation in joints. This fibrinolytic processresults in premature loss of the fibrin clot scaffold and disruption ofthe healing process for tissues within the joint or withinintra-articular tissues.

Enhancing healing of ligaments using growth factors has been an area ofgreat interest and research. While the majority of studies have focusedon the use of a single growth factor to stimulate healing, the naturalwound healing process is an orchestration of multiple growth factorsreleased by platelets and other cells over time. To try to reproducethis in the in vitro and in vivo environment, prior investigators havelooked at sustained release carriers and viral vectors for release ofthese cytokines over days or weeks, as well as examining applications ofmultiple growth factors. These studies have shown some additive effectsof applied combinations of growth factors on the wound healing ofligaments; however, even with advanced application techniques, thecombinations of growth factors, timing of release and concentration ofrelease make optimization of these systems difficult.

An alternative method recently used to stimulate healing of the anteriorcruciate ligament is the application of activated platelet-rich plasma(PRP). PRP is a combination of the extracellular matrix proteinsnormally found in plasma (including fibrinogen and fibronectin) andplatelets. When platelets are activated by the exposed collagen of aligament injury, they begin to aggregate and release multiple growthfactors including platelet-derived growth factor (PDGFαα, PDGF αβ, PDGFββ), transforming growth factors-β (TGFβ1, TGFβ2), vascular endothelialgrowth factor, basic fibroblast growth factor (FGF2), IGF-1 andepithelial growth factor. Growth factor release typically occursimmediately upon platelet activation and is sustained at much lowerlevels for the life-span of the platelet—up to 5-7 days.

PRP can be used to increase local concentrations of active PDGF-αβ andTGF-β1 by over 300% when platelets are concentrated in the plasma to asimilar degree. This degree of platelet concentration can beaccomplished by several available systems. As seen in vivo, these levelsof cytokines released locally by these platelet concentrates can resultin increased fibroblast DNA synthesis and up-regulation of type Icollagen production and changes in collagen organization, and indeed theuse of far lower concentrations (10 ng/ml TGF-β1 and 20 ng/ml PDGF-αβcan influence fibroblast proliferation, fibroblast chemotaxis, collagenproduction and collagen organization. The use of PRP over purifiedgrowth factor concentrates provides the added benefit of additional ECMproteins which also stimulate cellular adhesion and collagen synthesis,particularly in the presence of collagen fibrils.

SUMMARY OF THE INVENTION

The invention relates in some aspects to methods and products thatfacilitate anterior cruciate ligament regeneration or healing.

In some aspects the invention is a composition of a sterile solution ofsolubilized collagen in a concentration of greater than 5 and less thanor equal to 50 mg/ml and having a viscosity of 1,000-200,000 centipoise,hydroxyproline in a concentration of 0.1-5.0 μg/ml, a neutralizing agentwherein the solution has an osmolarity of 280-350 mOs/kg, wherein thecomposition is free of thrombin.

In other aspects the invention is a composition of a sterile solution ofsolubilized collagen in a concentration of greater than 1 and less than5 mg/ml and having a viscosity of 1,000-200,000 centipoise,hydroxyproline in a concentration of 0.1-5.0 μg/ml, wherein the solutionhas an osmolarity of 280-350 mOs/kg, wherein the composition is free ofthrombin.

A dried powder composition of sterile solubilized collagen, at least oneof decorin and biglycan, and buffer salts, wherein the composition isfree of thrombin may be provided according to other aspects of theinvention.

In other aspects the invention is a quick set composition of a sterilesolution of solubilized collagen in a concentration of greater than 5and less than or equal to 50 mg/ml and having a viscosity of1,000-200,000 centipoises and a pH of 6.8-8.0, wherein the solution hasan osmolarity of 280-350 mOs/kg, wherein the solution sets into ascaffold within 10 minutes of exposure to temperatures of greater than30° C. The solution in some embodiments may be a liquid or a gel.

In some embodiments the composition further comprises a buffer. Thecomposition may have a pH of 6.8-8.0. In some embodiments thecomposition has a pH of 7.4. In some embodiments the solution ismaintained at a temperature of 4° C.

In some embodiments the solubilized collagen is present in aconcentration of greater than 15 mg/ml. The collagen may be Type I, IIor III collagen in some embodiments. The collagen may be pepsinsolubilized collagen, enzyme solubilized collagen or it may beatelocollagen in certain embodiments.

In some embodiments each of the compositions includes at least one ofdecorin and biglycan. In other embodiments each of the compositionincludes both decorin and biglycan.

The composition may include other components, such as, an antibiotic, ananti-plasmin agent, a plasminogen activator inhibitor, fibrinogen, aglycosaminoglycan, insoluble collagen, a non-toxic cross-linking agent,or an accelerator. The composition may also include platelets or whiteblood cells. In other embodiments, the composition may include aneutralizing agent.

In other aspects, the invention is a method for preparing a collagenscaffold, by preparing a sterile solution of solubilized collagen in aconcentration of greater than 5 and less than or equal to 50 mg/ml andhaving a viscosity of 1,000-200,000 centipoises, and subjecting thesterile solution of solubilized collagen to a temperature of at least30° C. wherein the sterile solution of solubilized collagen forms acollagen scaffold.

In some embodiments, the collagen scaffold includes any of the optionalcomponents or has any of the properties described above.

A method for preparing a collagen scaffold by preparing a sterilesolution of solubilized collagen in a concentration of greater than 1and less than 5 mg/ml and having a viscosity of 1,000-200,000centipoises, and subjecting the sterile solution of solubilized collagento a temperature of at least 30° C. wherein the sterile solution ofsolubilized collagen forms a collagen scaffold is provided according toother aspects of the invention.

In some embodiments, the collagen scaffold includes any of the optionalcomponents or has any of the properties described above. In otherembodiments the collagen scaffold includes an accelerator.

In other aspects a kit, including a first container housing asolubilized collagen solution in a concentration of greater than 5 andless than or equal to 50 mg/ml and having a viscosity of 1,000-200,000centipoise, buffer salts housed in the first container or in a secondcontainer, and instructions for preparing a solution from thesolubilized collagen solution and the buffer salts is provided.

A kit, including a container housing a solubilized collagen solution ina concentration of greater than 5 and less than or equal to 50 mg/ml andhaving a viscosity of 1,000-200,000 centipoise, a device for housingblood, and instructions for preparing a gel from the solubilizedcollagen solution and blood components isolated from the blood housed inthe device is provided according to other aspects of the invention.

In another aspect, the invention is a kit, including a container housinga powder comprising collagen, a device for housing blood, andinstructions for preparing a gel from the solubilized collagen solutionand blood components isolated from the blood housed in the device. Inone embodiment the powder includes a neutralization agent.

In some embodiments, the collagen scaffold includes any of the optionalcomponents or has any of the properties described above. For instance,in some embodiments the solution is a liquid or a gel.

In certain embodiments the buffer salts are housed in the firstcontainer and are part of the solubilized collagen solution. In otherembodiments the buffer salts are housed in the second container.

The kit may also include a container housing a neutralization solution.

The kit may also include a device for housing blood. In some embodimentsthe device for housing the blood is a syringe that is capable of beingused for collecting blood. In other embodiments the device for housingthe blood is a centrifuge tube. An anticoagulant may optionally beincluded in the device for housing the blood or in a separate container.In yet other embodiments the kit includes a vortex tube.

The invention according to other aspects is a method comprisingcontacting the ends of a ruptured articular tissue in a subject with asterile solution of solubilized collagen in a concentration of greaterthan 5 and less than or equal to 50 mg/ml and having a viscosity of1,000-200,000 centipoises and a pH of 6.8-8.0, and hydroxyproline in aconcentration of 0.1-5.0 μg/ml, wherein the solution has an osmolarityof 280-350 mOs/kg, wherein the composition does not include thrombin,and allowing the solution to set to treat the ruptured articular tissue.

In some embodiments the articular tissue is intra-articular tissue. Anintra-articular injury may be, for instance, a meniscal tear, ligamenttear or a cartilage lesion.

In other embodiments the articular tissue is extra-articular tissue. Anextra-articular injury may be, for instance, ligament, tendon or muscleinjury.

The method may involve mechanically joining the ends of the rupturedtissue.

A method for replacing a ruptured articular tissue, by mechanicallysecuring a prosthetic device to tissue proximal to a site of rupturedarticular tissue, wherein the prosthetic device has an inductive coreand an adhesive zone disposed on at least a portion of the inductivecore and which is adapted to provide a microenvironment between thetissue proximal to a site of ruptured articular tissue and the inductivecore to promote cell migration from the tissue proximal to a site ofruptured articular tissue into the inductive core; and allowing bonds toform between the tissue proximal to a site of ruptured articular tissueand the adhesive zone of the prosthetic device is provided according toother aspects of the invention. in one embodiment the tissue proximal toa site of ruptured articular tissue is bone. In another embodiment theinductive core is a collagen sponge.

In another aspect, the invention is a method comprising contacting theends of a ruptured articular tissue in a subject with a sterile solutionof solubilized collagen and white blood cells in a concentration of atleast 4×10³ wbc/ml, and allowing the solution to set to treat theruptured articular tissue.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways. Also, the phraseology and terminology usedherein is for the purpose of description and should not be regarded aslimiting. The use of “including”, “comprising”, or “having”,“containing”, “involving”, and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures are illustrative only and are not required for enablement ofthe invention disclosed herein.

FIG. 1 is a graph depicting release of PDGF-αβ over time from bovinethrombin-activated (BT) and collagen-activated (CENTR (centrifuged PRP),PC (platelet concentrate) and RBC Reduced platelet concentrate) PRPhydrogels.

FIG. 2 is a graph depicting release of TGF-β1 over time from bovinethrombin-activated (BT) and collagen-activated (CENTR, PC and RBCReduced platelet concentrate) PRP hydrogels.

FIG. 3 is a graph depicting TGF-β1 release as a function of plateletconcentration in the PRP at 12 hours after platelet activation

FIG. 4 is a graph depicting PDGF-αβ release from the PRP gels as afunction of platelet concentration in the PRP at 12 hours after plateletactivation.

FIG. 5 is a graph depicting PDGF-αβ elution over time from thecell-seeded PRP hydrogels. The negative values over time suggestcell-based consumption of the PDGF-αβ.

FIG. 6 is a graph depicting VEGF elution over time from the cell-seededPRP hydrogels. The positive trend over time suggests continuing greaterproduction than consumption of the VEGF by the ACL cells.

FIG. 7 is a graph depicting cellular proliferation within the gels.

FIG. 8 is a graph depicting results of ACL cell-mediated gelcontraction.

FIG. 9 is a graph depicting results of in vivo pig total ACL transectiontreated with collagen slurry/buffer mixed with animal's own PRP in theoperating room, wherein the mixture is injected into the gap between thecut ligament ends.

FIG. 10 is a graph depicting cell number as a function of time inculture and collagen concentration.

FIG. 11 is a scan of an SDS-PAGE gel depicting the components of acollagen solution of the invention.

FIG. 12 is a graph depicting VEGF release as a function of granulocyteconcentration in the PRP at 12 hours after platelet activation.

FIG. 13A: Mean elastic modulus for the collagen-PRP hydrogels as afunction of mixing time.

represents a statistically significant difference between 30 seconds and120 seconds.

represents a statistically significant difference between 60 seconds and120 seconds. Error bars represent ±one standard deviation.

FIG. 13B: Mean inelastic modulus for the collagen-PRP hydrogels as afunction of mixing time.

represents a statistically significant difference between 30 seconds and120 seconds.

represents a statistically significant difference between 60 seconds and120 seconds. Error bars represent ±one standard deviation. good

FIG. 14A: Mean elastic modulus for the collagen-PRP hydrogels as afunction of mixing speed. The differences between the three groups werenot statistically significant. Error bars represent ±one standarddeviation.

FIG. 14B: Mean inelastic modulus for the collagen-PRP hydrogels as afunction of mixing speed. The differences between the three groups werenot statistically significant. Error bars represent ±one standarddeviation.

FIG. 15A: Mean elastic modulus for the collagen-PRP hydrogels as afunction of heating rate. The differences between the groups were notstatistically significant. Error bars represent ±one standard deviation.

FIG. 15B: Mean inelastic modulus for the collagen-PRP hydrogels as afunction of mixing speed. The differences between the groups were notstatistically significant. Error bars represent ±one standard deviation.

FIG. 16A: Mean elastic modulus for the collagen-PRP hydrogels as afunction of injection temperature.

represents a statistically significant difference between 24° C.-26° C.and all other groups.

represents a statistically significant difference between 26° C.-28° C.and all other groups. Error bars represent ±one standard deviation.

FIG. 16B: Mean inelastic modulus for the collagen-PRP hydrogels as afunction of injection temperature.

represents a statistically significant difference between 24° C.-26° C.and all other groups.

represents a statistically significant difference between 26° C.-28° C.and all other groups. Error bars represent ±one standard deviation.

FIG. 17: Mean time to 45° for the collagen-PRP hydrogels as a functionof injection temperature.

represents a statistically significant difference between 24° C.-26° C.and all other groups.

represents a statistically significant difference between 26° C.-28° C.and all other groups. ♦ represents a statistically significantdifference between 28° C.-30° C. and all other groups. Error barsrepresent ±one standard deviation.

FIG. 18 is a graph depicting in vivo results of failure strength versustemperature at injection.

FIG. 19: Coronal (A) view of a caprine knee with an intact ACL. Theblack arrows designate the ACL itself.

FIG. 20: Graft healing in the collagen group (sagittal view). The graftappears similar to the appearance at implantation (white arrow);however, there is scar mass present behind the graft (black arrow).

FIG. 21A and FIG. 21B: Graft healing in the collagen-platelet group. Thegraft is larger than at implantation and appears grossly to besynovialized and infiltrated with fibrovascular tissue. Good integrationwas observed at the insertion sites. (21A=coronal view, 21B=sagittalview).

FIG. 22 is a graph depicting strength of the joint as a function ofplatelet count.

FIGS. 23A and 23B are bivariate scattergrams wit regression 95%confidence bands.

FIG. 23A depicts fail load as a function of platelet count. FIG. 23Bdepicts stiffness as a function of platelet count.

FIG. 24: AP laxity jig assembled in Instron Machine. The femoral shaftis secured in the upper fixture which can be rotated to place the kneebetween 0 and 90 degrees of flexion for testing. All testing in thisexperiment was performed with the knee at 60 degrees of flexion.

FIG. 25: Sample graph of the AP laxity testing load versus displacementdata. This test was for the sutures tied through both the anterior andmiddle tunnels with the knee flexed 30 degrees. The resulting AP laxityis 8.7 mm, measured as the distance on the x axis between the twovertical regions of the curve.

FIG. 26: Anatomy of the ACL insertion in the porcine knee. In the pig,there are two discrete tibial insertion sites of the ACL—one isposterolateral, behind the anterior horn attachment of the medialmeniscus, and the second is anteromedial, located between the anteriorhorn attachment of the medial meniscus and the anterior horn attachmentof the lateral meniscus (forceps are retracting the anterior hornattachment of the lateral meniscus).

FIG. 27A, FIG. 27B and FIG. 27C: Photographs of the Anterior, Middle andPosterior tibial tunnel positions. The exit site of a Hewson suturepasses through the three sites respectively.

FIG. 28: Individual values for each of the six knees for the varioustesting positions. The intact knee laxity (column 1) is best restored inthe groups where sutures passed through the anterior or middle tunnelsand tied with the knee in 60 degrees of flexion (columns 6, 8 and 12).

FIG. 29: Differences from the intact AP laxity of the knee for allrepair conditions. Bars represent the mean, error bars represent thestandard error of the mean. N=6 for all groups. Groups not significantlydifferent from intact are marked by an asterisk.

FIG. 30 is a graph depicting cell counts within the sponge/PRPpreparations measured at Day 2 and Day 10.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the invention relate to compositions and methods forrepairing damaged articular tissue. The invention involves novelcollagen based compositions and formulations for repairing articulartissue, such as a ruptured or torn ligament for instance. Thecompositions may be used alone or in combination with three-dimensional(3-D) scaffolds or other traditional repair devices. The materialprovides a connection between the ruptured ends of the ligament andfibers, or provides a replacement, alone or in combination with otherdevices, for a torn ligament, after injury, and encourages the migrationof appropriate healing cells to form scar and new tissue, thusfacilitating healing and regeneration.

It is intended that the use of the compositions and methods of thepresent invention involve the repair, replacement, reconstruction oraugmentation of specific tissue types. Articular injuries include bothintra-articular and extra-articular injuries. Intra-articular injuriesinvolve, for instance, injuries to meniscus, ligament and cartilage.Extra-articular injuries include, but are not limited to injuries to theligament, tendon or muscle. Thus, the methods of the invention may beused to treat injuries to the Anterior cruciate ligament (ACL), Lateralcollateral ligament (LCL), Posterior cruciate ligament (PCL), Medialcollateral ligament (MCL), Volar radiocarpal ligament, Dorsalradiocarpal ligament, Ulnar collateral ligament, Radial collateralligament, meniscus, labrum, for example glenoid labrum and acetabularlabrum, cartilage, for example, and other tissues exposed to synovialfluid after injury.

The injury being treated may be, for instance, a torn or rupturedligament. A ligament is a short band of tough fibrous connective tissuecomposed of collagen fibers. Ligaments connect bones to other bones toform a joint. A torn ligament is one where the ligament remainsconnected but has been damaged causing a tear in the ligament. The tearmay be of any length or shape. A ruptured ligament is one where theligament has been completely severed providing two separate ends of theligament. A ruptured ligament may provide two ligament ends of similaror different lengths. The rupture may be such that a ligament stump isformed at one end.

An example of a ruptured anterior cruciate ligament is described forexemplary purposes only. The anterior cruciate ligament (ACL) is one offour strong ligaments that connects the bones of the knee joint. Thefunction of the ACL is to provide stability to the knee and minimizestress across the knee joint. It restrains excessive forward movement ofthe lower leg bone, the tibia, in relation to the thigh bone, the femur,and limits the rotational movements of the knee. An anterior cruciateligament is ruptured such that it no longer forms a connection betweenthe femur bone and the tibia bone. The resulting ends of the rupturedACL may be of any length. The ends may be of a similar length, or oneend may be longer in length than the other.

The repair of the damaged tissue is achieved using collagen based repairmaterial alone or in combination with a tissue healing device. A tissuehealing device is a device other than the repair material that aids inthe repair of the damaged tissue and includes, for instance, scaffolds,such as sponges and grafts and mechanical devices, such as sutures andanchors.

The damaged or injured tissue is treated with a novel composition whichis a sterile solution of solubilized collagen. Solubilized collagen, asused herein, is enzyme solubilized collagen including one or more ofType I, II, III, IV, V, X collagen. Preferably the enzyme solubilizedcollagen is tropocollagen or Atelocollagen rather than fibrillarcollagen in order to reduce the antigenicity of the material. Thecollagen is isolated from a source and mechanically minced and broken upin an enzyme based acid media rather than aqueous or salt solution. Forinstance, the collagen may be solubilized in pepsin. The step ofmechanically mincing the collagen is important for homogenization toproduce a material of uniform consistency that is free of aggregates andlumps.

The pH of the solution during the solubilization is very acidic, forinstance, a pH=2.0 is normally obtained during solubilization withpepsin. A preferred pH for storage of the material is 2.0 to 6.5.Preferably the collagen is kept cold (4° C. or on ice) during storageand throughout the preparation.

In one embodiment the solubilized collagen is Type I collagen. As usedherein the term, “Type I collagen” is characterized by two α1(I) chains,and one α2(I) chains (heterotrimeric collagen). The α1 (I) chains areapproximately 300 nm long. Type I collagen is predominantly found inbone, skin (in sheet-like structures), and tendon (in rope-likestructures). Type I collagen is further typified by its reaction withthe protein core of another connective tissue component known as aproteoglycan. Type I collagen contains signaling regions that facilitatecell migration.

The collagen is synthetic or naturally derived. Natural sources ofcollagen may be obtained from animal or human sources. For instance, itmay be derived from rat, pig, cow, or human tissue or tissue from anyother species. Tendons, ligaments, muscle, fascia, skin, cartilage,tail, or any source of collagenous tissue are useful. The material isthen implanted into a subject of the same or different species. Theterms “xenogeneic” and “xenograft” refer to cells or tissue whichoriginates with or is derived from a species other than that of therecipient. Alternatively the collagen may be obtained from autologouscells. For instance, the collagen may be derived from a patient'sfibroblasts which have been cultured. The collagen may then be used inthat patient or other patients. The terms “autologous” and “autograft”refer to tissue or cells which originate with or are derived from therecipient, whereas the terms “allogeneic” and “allograft” refer to cellsand tissue which originate with or are derived from a donor of the samespecies as the recipient. The collagen may be isolated anytime beforesurgery.

The solubilized collagen may be in a concentration of 1-50 mg/ml in thesolution. In some embodiments that concentration of solubilized collagenis greater than 5 mg/ml and less than or equal to 50 mg/ml. Theconcentration of collagen may be, for instance, 10, 15, 20, 25, 30, 35,or 40 mg/ml. Such high concentrations of collagen are useful forproducing viscosity levels that are desirable for the methods of theinvention. Most commercially available collagen solutions are of lowerconcentrations. Higher concentrations can be made, for instance, usingthe methods described herein. In other embodiments the solubilizedcollagen solution has a concentration of 1 mg/ml to less than 5 mg/ml.When such lower concentrations of collagen are used, additionalcomponents or steps are taken to increase the viscosity of the materialin order to be useful according to the methods of the invention.Examples of viscosity inducing methods or components are describedherein.

The solution should be prepared, by varying the collagen content andother components, to provide the desired flow properties of the finishedcomposition. In some embodiments the solution has a collagen viscosityof 1,000 to 200,000 centipoise.

The collagen solution is sterile for in vivo use. The solution may besterilized and/or components of the solution may be isolated understerile conditions using sterile techniques to produce a sterilecomposition. The final desired properties of the composition may bedeterminative of how the solution is sterilized because somesterilization techniques may affect properties such as viscosity. Ifcertain components of the solution are not to be sterilized, i.e., thecollagen isolated from natural sources, the remaining components can becombined and sterilized before addition of the collagen, or eachcomponent can be sterilized separately. The solution can then be made bymixing each of the sterilized components with the collagen that has beenisolated using sterile techniques under sterile conditions.Sterilization may be accomplished, for instance, by autoclaving attemperatures on the order of about 115° C. to 130° C., preferably about120° C. to 125° C. for about 30 minutes to 1 hour. Gamma radiation isanother method for sterilizing components. Filtration is also possible,as is sterilization with ethylene oxide.

The solubilized collagen solution may contain additional components,such as insoluble collagen, other extracellular matrix proteins (ECM),such as proteoglycans and glycosaminoglycans, fibronectin, laminin,entectin, decorin, lysyl oxidase, crosslinking precursors (reducible andnon-reducible), elastin, elastin crosslink precursors, cell componentssuch as, cell membrane proteins, mitochondrial proteins, nuclearproteins, cytosomal proteins, and cell surface receptors, growthFactors, such as, PDGF, TGF, EGF, and VEGF, and hydroxyproline. In someembodiments hydroxyproline may be present in the solution in aconcentration of 1 to 3.0 m/ml, which may be 8 to 9% of the totalprotein in the collagen solution. In some embodiments, thehydroxyproline is present in a concentration of 0.5 to 4.0 μg/ml in thecollagen solution prior to the addition of any buffer. In someembodiments the collagen solution is free of thrombin. “Free ofthrombin” as used herein refers to a composition which has less than 1%thrombin. In some embodiments, free of thrombin refers to undetectablelevels. In other embodiments it refers to 0% thrombin.

The collagen is mixed with one or more buffers to produce a solutionhaving a desirable pH range for subsequent mixing with cells andapplication to the body. Ideally the buffer solution(s) has no toxiccomponents or residue, confers physiologic osmolarity and has thecapacity to keep the solution at physiologic pH. A preferred buffer usedin accordance with the invention is a HEPES buffer. However, any bufferthat is non-toxic and is capable of regulating the pH and/or osmolarityto the levels described herein is useful according to the invention.HEPES is N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid, (molecularweight and structure=238.31, C₈H₁₈N₂O₄S). For instance, the inventorshave found the following buffer solution achieves the appropriate pH andosmolartiy values:

-   -   0.1M HEPES    -   10× Ham's F-10 medium    -   100× antibiotic/antimycotic solution (10,000 I.U. Penicillin,        10,000 μg/mL Streptomycin, 25 μg/mL Amphotericin B from CellGro        by Mediatech)    -   Ultra pure sterile water    -   7.5% sodium bicarbonate    -   NaHCO₃

The components of 10× Ham's F-10 include the following:

Formulation (as 10×):

Mol. Mol. Component mg/lt Wt. (mM) Amino Acids L-Alanine 89.10000 89.11.00 L-Arginine HCl 2107.00000 174.2 12.10 L-Asparagine H₂O 150.10000150.1 1.00 L-Aspartic Acid 133.10000 133.1 1.00 L-Cysteine HCl H₂O351.30000 175.6 2.00 L-Glutamic Acid 147.10000 147.1 1.00 Glycine75.10000 75.07 1.00 L-Histidine HCl H₂O 209.60000 209.6 1.00L-Isoleucine 26.20000 131.2 0.20 L-Leucine 131.20000 131.2 1.00 L-LysineHCl 293.00000 182.6 1.60 L-Methionine 44.80000 149.2 0.30L-Phenylalanine 49.60000 165.2 0.30 L-Proline 115.10000 115.1 1.00L-Serine 105.10000 105.1 1.00 L-Threonine 35.70000 119.1 0.30L-Tryptophan 6.10000 204.2 0.03 L-Tyrosine 18.10000 181.2 0.10 L-Valine35.10000 117.1 0.30 Vitamins Biotin 0.24000 244.3 0.0010 CholineChloride 6.98000 139.6 0.05 D-Calcium Pantothenate 7.15000 238.3 0.03Folic Acid 13.20000 441.4 0.03 myo-Inositol 5.41000 180.2 0.03Nicotinamide 6.11000 122.13 0.05 Pyridoxine HCl 2.06000 205.6 0.01Riboflavin 3.76000 376.4 0.01 Thiamine HCl 10.12000 337.3 0.03 VitaminB12 13.60000 1355.4 0.01 Inorganic Salts Calcium Chloride [CaCl₂ 2H₂O]441.00000 147 3.00 Dihydrate Cupric Sulfate [CuSO₄] 0.01600 159.680.0001 Ferrous Sulfate [FeSO₄ 7H₂O] 8.34 278 0.03 Heptahydrate MagnesiumSulfate [MgSO₄] 746.00000 120.4 6.20 Potassium Chloride [KCl] 2850.0000074.55 38.23 Potassium Phosphate Monobasic 830.00000 136.09 6.10 [KH₂PO₄]Sodium Chloride [NaCl] 74000.00000 58.44 1266.26 Sodium PhosphateDibasic 1562.00000 141.96 11.00 [Na₂HPO₄] Zinc Sulfate [ZnSO₄ 7H₂O]0.28800 287.5 0.0010 Heptahydrate Other Dextrose 11000.00000 180.2 61.04Hypoxanthine 40.80000 136.1 0.30 Lipoic Acid 2.06000 206.3 0.01 PhenolRed Sodium Salt 12.40000 376.4 0.03 Sodium Pyruvate 1100.00000 110 10.00Thymidine 7.27000 242.2 0.03

The above-described buffer is exemplary. Many of the components are notessential For instance, it is not essential to use sterile water, aslong as the appropriate osmolarity is maintained. The 10× F10 solutionis also optional. The buffer may be prepared without 10× F10 orequivalent solution. Additionally glucose or other sugar may be used inplace of the 10× F10.

The buffer may or may not include an antibiotic. For instance, theantibiotic may be penicillin/streptomycin as described above.Alternatively it may be a clinical antibiotic, which is used in humanpatients for the treatment or prevention of diseases, such as any ofthose described in Remington's Pharmaceutical Sciences (Mack PublishingCo., Easton Pa.), which is hereby incorporated by reference.

The buffer may be a single component or it may be multiple componentsadded at the same time or different times. If the buffer is a singlecomponent it should have properties that enable it to produce a solutionhaving a desirable pH range and osmolarity. In some instances it isdesirable to have at least two buffer components, a collagen buffersolution and a neutralizing buffer. The collagen buffer solution may beused to prepare the collagen in a solution. In some instances theprepared collagen solution may be stored for extended periods of time.

A neutralizing buffer, also referred to as a neutralizing agent, may beadded as a solution or in the form of dried salts to a collagensolution. Once the neutralizing buffer is added the solution should bekept cold. If the materials are being processed at room temperature forextended periods of time, it is preferred that the neutralizing bufferbe added to the collagen solution after storage. Thus, a collagensolution without a neutralization agent may be prepared ahead of timeand stored or it may be prepared during surgery and used immediately.The neutralization agent may be added at surgery or ahead of time, but aneutralized collagen solution preferably should be kept cold (4° C. oron ice).

After the neutralizing agent is added to the collagen solution anosmolarity of 250 to 350 mOsm/kg is preferably achieved. Osmolarity is acount of the total number of osmotically active particles in a solutionand is equal to the sum of the molarities of all the solutes present inthat solution. It is defined as a measure of the osmoles of solute perlitre of solution. Osmolarity is a measure of the osmoles of solute perkilogram of solvent. One of skill in the art can determine theosmolarity of a solution by obtaining measurements using an osmometer.An equation used to determine the osmolarity of a solution is:

Osm=φnC

wherein

-   -   φ is the osmotic coefficient and accounts for the degree of        dissociation of the solute. φ is between 0 and 1 where 1        indicates 100% dissociation.    -   n is the number of particles into which a molecule dissociates.    -   C is the molal concentration of the solution

Additionally, after the neutralizing agent is added, preferably a pHbetween 6.8 and 9.0 is achieved. In some embodiments a pH of 6.8-8.0 ispreferred. In other embodiments a pH of 7.2-7.6 or even 7.4 ispreferred.

Preferably the buffer is sterile prior to addition to the collagensolution. If it is unsterile then it should be sterilized prior toaddition to the collagen solution or the whole collagen/buffer solutionshould be sterilized as described herein. The components of the buffermay be unsterile and then filtered at a point before it is mixed withthe collagen.

In certain embodiments, the collagen solution is mixed with cells suchas platelets or white blood cells. In some embodiments, the cells arederived from the subject to be treated. In other embodiments, the cellsare derived from a donor that is allogeneic to the subject.

In certain embodiments, platelets may be obtained as platelet richplasma (PRP). This component contains fibrin and platelets as well asother plasma proteins found in the blood. There may also be some whiteblood cells (WBC) and red blood cells (RBC) found in this preparation.Preferably the platelet concentration of PRP is at least 100K/ml, andpreferably over 300K/ml. For instance, the platelet concentration may beat least 1× what it is in the blood of the patient, and preferably 1.5×or greater In order to maintain the stability of the cells a physiologicpH (i.e., 6.2 to 7.6) and a physiologic plasma osmolarity (i.e., 280-360osms/kg) is used. In order to enhance the function of the PRP,preferably the PRP is used within 7 days of being drawn from the patientor donor. Often the PRP is isolated from the patient at time of surgery.Preferably it is stored at 20 to 24° C. (room temp). However, isolationand storage of the cells may be achieved by any methods and for anylength of time known in the art for maintaining the activity of theactive components.

In a non-limiting example, platelets may be isolated from a subject'sblood using techniques known to those of ordinary skill in the art. Asan example, a blood sample may be centrifuged at 700 rpm for 20 minutesand the platelet-rich plasma upper layer removed. Platelet density maybe determined using a cell count as known to those of ordinary skill inthe art. The platelet rich plasma may be mixed with collagen and appliedto the patient.

In a non-limiting example, white blood cells may also be isolated from asubject's blood using techniques known to those of ordinary skill in theart. As an example, a blood sample may be centrifuged at 700 rpm for 20minutes and the buffy coat containing white blood cells removed. WBCdensity may be determined using a cell count as known to those ofordinary skill in the art. The WBCs can be mixed with collagen andapplied to the patient.

The collagen solution may also include any one or more of ananti-plasmin agent, an extracellular matrix (ECM) protein, other proteinor enzyme inhibitors, antibodies to plasmin, antibodies to tissueplasminogen activator or urokinase plasminogen activator, non-toxiccrosslinkers, calcium, dextrose or other sugars and cell nutrients inphysiological concentrations. Anti-plasmin agents include but are notlimited to antifibrinolytic enzymes such as plasminogen inactivator,plasminogen binding α₂ antiplasmin, non-plasminogen binding α₂antiplasmin, α₂ macroglobulin, α₂ plasmin inhibitor, α₂ antiplasmin, andthrombin activatable fibrinolysis inhibitor. Other protein or enzymeinhibitors include but are not limited to anti-enzymatic proteinsincluding inhibitors of collagenase, trypsin, matrix metalloproteinases,elastase and hyaluronidase. The ECM is composed of fibrillar andnon-fibrillar components. The major fibrillar proteins are collagen andelastin. The ECM includes for instance, diverse combinations ofcollagens, fibrinogen, proteoglycans, elastin, hyaluronic acid, andvarious glycoproteins including laminin, fibronectin, heparan sulfateproteoglycan, and entactin. Non-toxic crosslinkers include but are notlimited to tissue transglutaminases, lysyl oxidase, fibrin, fibronectin,and reducible and non-reducible crosslink precursor molecules.

The collagen solution, with or without any of the above-describedadditional components, may be stored as a liquid or gel material or maybe dried and stored as a powder. For instance, a collagen solution maybe lyophilized to produce a powder. The powder may then be reconstitutedin a buffer solution. Neutralizing agent may be present in thereconstitution buffer or may be added as a separate buffer or as salts.

The final collagen solution includes collagen, buffer and cells, such asPRP or WBCs. The components are mixed on a microscopic level, ratherthan layered. Preferably it has a pH of 7.4 and a minimum viscosity ofapproximately 1,000 centipoise. Preferably the viscosity is in the rangeof 1,000-200,000 centipoise.

While the degree of “solidness” may vary from application toapplication, generally speaking collagen solutions of the presentinvention will exhibit viscosities in the full range of from liquid togel-like to solid-like. A collagen solution having optimal viscosity canbe obtained directly from the source of collagen, depending on theconcentration of the collagen. However, a collagen solution not havingan optimal viscosity can be manipulated to create the correct viscosity.The viscosity of a collagen solution may be lowered by diluting thesolution. The viscosity of a lower viscosity collagen solution may beincreased to increase gelation. Gelation is the change in viscosity froma fluid-like composition to a solid or gel-like composition. Gelation orviscosity of a solution may be increased by adding one or more of thefollowing: other ECM molecules, including but not limited to, insolublecollagen, fibrin, fibronectin, and cellulose; cell additions, includingbut not limited to, platelets and fibroblasts; non-toxic crosslinkingagents, including but not limited to, tissue transglutaminases, lysyloxidase, fibrin, and fibronectin; and other high viscosity materialswith low osmolarity, including but not limited to, alginate andsynthetic filler materials.

One example of a method for preparing and using the collagen solution ofthe invention is provided. The methods of the invention are not solimited and the description is provided for exemplary purposes only.Collagen is isolated from rat tails and processed 6 weeks to 6 monthsahead of application time (surgery). A buffer solution is mixed ahead oftime as well. The buffer is designed so collagen-buffer mixture willhave pH of 7.4 and be iso-osmotic with plasma. The PRP is obtained fromblood taken from the patient during anesthesia for surgery using a largebore needle and anticoagulant. The blood is centrifuged to get a PRPwith platelet count of at least 1× normal. When the surgical site isready, the collagen and buffer (containing neutralizing agent) are mixedfirst using vortex. The PRP is then added to the neutralizedcollagen-buffer mixture. The PRP and collagen are combined using amixing process to produce a repair material. The resultant gel isinjected arthroscopically into the joint wound site to promote thehealing process.

The term “repair material” as used herein refers to the finalformulation of collagen solution with cells to be delivered to thesubject.

The collagen solution or repair material may include additionalmaterials, such as growth factors, antibiotics, insoluble or solublecollagen, a cross-linking agent, thrombin, stem cells, a geneticallyaltered fibroblast, platelets, water, plasma, extracellular proteins anda cell media supplement. Alternatively the collagen solution or repairmaterial may exclude any of these components, and in particularthrombin. The additional materials may be added to affect cellproliferation, extracellular matrix production, consistency, inhibitionof disease or infection, tonicity, cell nutrients until nutritionalpathways are formed, and pH of the collagen solution or repair material.All or a portion of these additional materials may be mixed with thecollagen solution or repair material before or during implantation, oralternatively, the additional materials may be implanted proximate tothe defect area after the repair material is in place.

In general, the collagen solution is prepared in advance or at the timeof surgery. At a temperature of preferably 4° C. to around roomtemperature PRP or WBCs are added. The PRP/WBC collagen mixture is kepton ice until use. Just prior to use the mixture may be warmed to atemperature of 24-30° C. (preferably 28° C.) and then immediatelyinjected into the subject. In the subject the material is subjected tobody temperatures in excess of 30 C to produce a gel.

The repair material of the invention, as discussed above, may be applieddirectly to the tissue alone or it may be used in combination with atissue healing device such as a scaffold. Scaffolds may be synthetic ornaturally occurring, such as in a graft. A device or scaffold may be anyshape that is useful for implantation into a subject. The scaffold, forinstance, can be tubular, semi-tubular, cylindrical, including either asolid cylinder or a cylinder having hollow cavities, a tube, a flatsheet rolled into a tube so as to define a hollow cavity, liquid, anamorphous shape which conforms to that of the repair space, a “Chinesefinger trap” design, a trough shape, or square. Other shapes suitablefor the scaffold of the device as known to those of ordinary skill inthe art are also contemplated in the invention.

The scaffold may be pretreated with the repair material prior toimplantation into a subject. For instance, the scaffold may be soaked ina repair material prior to or during implantation into a repair site.The repair material may be injected directly into the scaffold prior toor during implantation. The repair material may be injected within ascaffold at the time of repair.

A scaffold is capable of insertion into a repair site and either forminga connection between the ends of a ruptured tissue, or forming around atorn tissue such that, in either case, the integrity and structure ofthe tissue is maintained. A scaffold is preferably made of acompressible, resilient material which has some resistance todegradation by synovial fluid. Synovial fluid as part of normal jointactivity, naturally prevents clot formation. This fibrinolytic processwould result in the premature degradation of the scaffold and disruptthe healing process of the tissue. The material may be natural orsynthetic and may be either permanent or biodegradable material, such aspolymers and copolymers. The scaffold can be composed, for example, ofcollagen fibers, collagen gel, foamed rubber, natural material,synthetic materials such as rubber, silicone and plastic, ground andcompacted material, perforated material, or a compressible solidmaterial.

A scaffold that is capable of compression and expansion is particularlydesirable. For example, a sponge scaffold may be compressed prior to orduring implantation into a repair site. A compressed sponge scaffoldallows for the sponge scaffold to expand within the repair site.Examples of scaffolds useful according to the invention are found inU.S. Pat. No. 6,964,685 and US Patent Application Nos. 2004/0059416 and2005/0261736, the entire contents of each are herein incorporated byreference.

An important subset of natural matrices are those made predominantlyfrom collagen, the main structural component in ligament. Collagen canbe of the soluble or the insoluble type. Preferably, the collagen issoluble, e.g., acidic or basic. For example, the collagen can be type I,II, III, IV, V, IX or X. Preferably the collagen is type I. Morepreferably the collagen is soluble type I collagen. Type I collagen isthe predominant component of the extracellular matrix for the humananterior cruciate ligament and provides an example of a choice for thebasis of a bioengineered scaffold. Collagen occurs predominantly in afibrous form, allowing design of materials with very differentmechanical properties by altering the volume fraction, fiberorientation, and degree of cross-linking of the collagen. The biologicproperties of cell infiltration rate and scaffold degradation may alsobe altered by varying the pore size, degree of cross-linking, and theuse of additional proteins, such as glycosaminoglycans, growth factors,and cytokines. In addition, collagen-based biomaterials can bemanufactured from a patient's own skin, thus minimizing the antigenicityof the implant (Ford et al., 105 Laryngoscope 944-948 (1995)).

Numerous matrices made of either natural or synthetic components havebeen investigated for use in tissue repair and reconstruction. Naturalmatrices are made from processed or reconstituted tissue components(such as collagens and GAGs). Because natural matrices mimic thestructures ordinarily responsible for the reciprocal interaction betweencells and their environment, they act as cell regulators with minimalmodification, giving the cells the ability to remodel an implantedmaterial, which is a prerequisite for regeneration.

Synthetic matrices are made predominantly of polymeric materials.Synthetic matrices offer the advantage of a range of carefully definedchemical compositions and structural arrangements. Some syntheticmatrices are not degradable. While the non-degradable matrices may aidin repair, non-degradable matrices are not replaced by remodeling andtherefore cannot be used to fully regenerate ligament. It is alsoundesirable to leave foreign materials permanently in a joint due to theproblems associated with the generation of wear particles, thusdegradable materials are preferred for work in regeneration. Degradablesynthetic scaffolds can be engineered to control the rate ofdegradation.

A scaffold may be a solid material such that its shape is maintained, ora semi-solid material capable of altering its shape and or size. Ascaffold may be made of expandable material allowing it to contract orexpand as required. The material can be capable of absorbing plasma,blood, other body fluids, liquid, hydrogel, or other material thescaffold either comes into contact with or is added to the scaffold.

A scaffold material can be protein, lyophilized material, or any othersuitable material. A protein can be synthetic, bioabsorbable or anaturally occurring protein. A protein includes, but is not limited to,fibrin, hyaluronic acid, or collagen. A scaffold material mayincorporate therapeutic proteins including, but not limited to,hormones, cytokines, growth factors, clotting factors, anti-proteaseproteins (e.g., alpha1-antitrypsin), angiogenic proteins (e.g., vascularendothelial growth factor, fibroblast growth factors), antiangiogenicproteins (e.g., endostatin, angiostatin), and other proteins that arepresent in the blood, bone morphogenic proteins (BMPs), osteoinductivefactor (IFO), fibronectin (FN), endothelial cell growth factor (ECGF),cementum attachment extracts (CAE), ketanserin, human growth hormone(HGH), animal growth hormones, epidermal growth factor (EGF),interleukin-1 (IL-1), human alpha thrombin, transforming growth factor(TGF-beta), insulin-like growth factor (IGF-1), platelet derived growthfactors (PDGF), fibroblast growth factors (FGF, bFGF, etc.), andperiodontal ligament chemotactic factor (PDLGF), for therapeuticpurposes. A lyophilized material is one that is capable of swelling whenliquid, gel or other fluid is added or comes into contact with it.

The repair material may also be used in combination with a scaffold thatis a graft, such as an ACL graft. Several types of ACL grafts areavailable for use by the surgeon in ACL reconstruction. The grafts maybe autografts that are harvested from the patient, for example patellarbone-tendon-bone grafts, or hamstring grafts. Alternatively, the graftscan be xenografts, allografts, or synthetic polymer grafts. Allograftsinclude ligamentous tissue harvested from cadavers and appropriatelytreated and disinfected, and preferably sterilized. Xenografts includeharvested connective tissue from animal sources such as, for example,porcine tissue. Typically, the xenografts must be appropriately treatedto eliminate or minimize an immune response. Synthetic grafts includegrafts made from synthetic polymers such as polyurethane, polyethylene,polyester and other conventional biocompatible bioabsorbable ornonabsorbable polymers and composites, such as the scaffolds describedherein.

Tissue healing devices also include mechanical devices such as suturesand anchors. An anchor is a device capable of insertion into a bone ortissue such that it forms a stable attachment to the bone or tissue. Insome instances the anchor is capable of being removed from the bone ifdesired. An anchor may be conical shaped having a sharpened tip at oneend and a body having a longitudinal axis. The body of an anchor mayincrease in diameter along its longitudinal axis. The body of an anchormay include grooves suitable for screwing the anchor into position. Ananchor may include an eyelet at the base of the anchor body throughwhich one or more sutures may be passed. The eyelet may be oval or roundand may be of any size suitable to allow one or more sutures to passthrough and be held within the eyelet.

An anchor may be attached to a bone or tissue by physical or mechanicalmethods as known to those of ordinary skill in the art. An anchorincludes, but is not limited to, a screw, a barb, a helical anchor, astaple, a clip, a snap, a rivet, or a crimp-type anchor. The body of ananchor may be varied in length. Examples of anchors, include but are notlimited to, IN-FAST™ Bone Screw System (Influence, Inc., San Francisco,Calif.), IN-TAC™ Bone Anchor System (Influence, Inc., San Francisco,Calif.), Model 3000 AXYALOOP™ Titanium Bone Anchor (Axya Medical Inc.,Beverly, Mass.), OPUS MAGNUM® Anchor with Inserter (Opus Medical, Inc.,San Juan Capistrano, Calif.), ANCHRON™, HEXALON™, TRINION™ (allavailable from Inion Inc., Oklahoma City, Okla.) and endobuttons andTwinFix AB absorbable suture anchor (Smith & Nephew, Inc., Andover,Mass.). Anchors are available commercially from manufacturers such asInfluence, Inc., San Francisco, Calif., Axya Medical Inc., Beverly,Mass., Opus Medical, Inc., San Juan Capistrano, Calif., Inion Inc.,Oklahoma City, Okla., and Smith & Nephew, Inc., Andover, Mass.

An anchor may be composed of a non-degradable material, such as metal,for example titanium 316 LVM stainless steel, CoCrMo alloy, or Nitinolalloy, or plastic. An anchor is preferably bioabsorbable such that thesubject is capable of breaking down the anchor and absorbing it.Examples of bioabsorbable material include, but are not limited to,MONOCRYL (poliglecaprone 25), PDS II (polydioxanone), surgical gutsuture (SGS), gut, coated VICRYL (polyglactin 910, polyglactin 910braided), human autograft tendon material, collagen fiber, POLYSORB,poly-L-lactic acid (PLLA), polylactic acid (PLA), polysulfone,polylactides (Pla), racemic form of polylactide (D,L-Pla),poly(L-lactide-co-D,L-lactide), 70/30 poly(L-lactide-co-D,L-lactide),polyglycolides (PGa), polyglycolic acid (PGA), polycaprolactone (PCL),polydioxanone (PDS), polyhydroxyacids, and resorbable plate material(see e.g. Orthopedics, October 2002, Vol. 25, No. 10/Supp.). The anchormay be bioabsorbed over a period of time which includes, but is notlimited to, days, weeks, months or years.

A suture is preferably bioabsorbable, such that the subject is capableof breaking down the suture and absorbing it, and synthetic such thatthe suture may not be from a natural source. Examples of suturesinclude, but are not limited to, VICRYL™ polyglactin 910, PANACRYL™absorbable suture, ETHIBOND® EXCEL polyester suture, PDS® polydioxanonesuture and PROLENE® polypropylene suture. Sutures are availablecommercially from manufacturers such as MITEK PRODUCTS division ofETHICON, INC. of Westwood, Mass.

A staple is a type of anchor having two arms that are capable ofinsertion into a bone or tissue. In some instances, the arms of thestaple fold in on themselves when attached to a bone or in someinstances when attached to other tissue. A staple may be composed ofmetal, for example titanium or stainless steel, plastic, or anybiodegradable material. A staple includes but is not limited to linearstaples, circular staples, curved staples or straight staples. Staplesare available commercially from manufacturers such as Johnson & JohnsonHealth Care Systems, Inc. Piscataway, N. J., and Ethicon, Inc.,Somerville, N.J. A staple may be attached using any staple device knownto those of ordinary skill in the art, for example, a hammer and staplesetter (staple holder).

The device may be inserted into a repair site of the ruptured or torntissue. A repair site is the area around a ruptured or torn tissue intowhich the material of the invention may be inserted. A device may beplaced into a repair site area during surgery using techniques known tothose of ordinary skill in the art. If a scaffold is used in themethods, the scaffold can either fill the repair site or partially fillthe repair site. A scaffold can partially fill the repair site wheninserted and expand to fill the repair site in the presence of blood,plasma or other fluids either present within the repair site or addedinto the repair site, such as the repair material.

The scaffold may be positioned in combination with a surgical technique.For instance, a hole may be drilled into a bone at or near a repair siteof a ruptured or torn tissue and the scaffold attached by a suturethrough the hole to the bone. A bone at or near a repair site is onethat is within close proximity to the repair site and can be utilizedusing the methods and devices of the invention. For example, a bone ator near a repair site of a torn anterior cruciate ligament is a femurbone and/or a tibia bone. A hole can be drilled into a bone using adevice such as a Kirschner wire (for example a small Kirschner wire) anddrill, or microfracture pics or awls.

A hole may be drilled into a bone on the opposite side to the repairsite. A suture may be passed through the hole in the bone and attachedto the bone. A scaffold is attached to the suture to secure the scaffoldbetween the bone and an end of a ruptured tissue. A ruptured tissueprovides two ends of the tissue that were previously connected. Ascaffold may be attached to one or both ends of a ruptured tissue by oneor more sutures. A suture may be attached to a second bone site at ornear the repair site. The suture may be attached to the second boneusing a second anchor.

In a typical arthroscopic procedure, for instance of the ACL, thesurgeon prepares the patient for surgery by insufflating the patient'sknee with sterile saline solution. Several cannulas are inserted intothe knee and used as entry portals into the interior of the knee. Aconventional arthroscope is inserted through one of the cannulas so thatthe knee may be viewed by the surgeon remotely.

In surgical reconstruction of a tissue such as ACL the surgeon may drilla tibial tunnel and a femoral tunnel in accordance with conventionalsurgical techniques using conventional surgical drills and drill guides.A replacement anterior cruciate ligament graft is then prepared andmounted in the tibial and femoral tunnels, and secured usingconventional techniques and known devices in order to complete the kneereconstruction.

The repair material is applied to a subject. The application to thesubject involves surgical procedures. The following is an example of asurgical procedure which may be performed using the methods of theinvention. The affected extremity is prepared and draped in the standardsterile fashion. A tourniquet may be used if indicated. Theintra-articular lesion is identified and defined, the tissue ends arepretreated, either mechanically or chemically, and if a scaffold isbeing used, the scaffold is introduced into the tissue defect. If thescaffold has not been pre-soaked in the repair material or if morerepair material is desired, then the repair material is added to thescaffold. The scaffold may be reinforced by placement of sutures orclips. If no scaffold is used the tissue defect is coated directly withrepair material. The post-operative rehabilitation is dependent on thejoint affected, the type and size of lesion treated, and the tissueinvolved.

The temperature of the repair material may be regulated to optimizerapid gelatin in vivo. For instance, it is shown in the examples thatdifferent temperatures at the time of injection of the repair materialinto the body can influence the time required for gelatin to occur. Insome embodiments of the invention the injection temperature is ideallybetween 24° C. and 30° C. 28° C. may be an optimal temperature in somesettings to cause the quickest gelatin time. The injection temperaturecan be achieved by warming the solution to the optimal temperatureimmediately prior to injection.

The methods of the invention may be achieved using arthroscopicprocedures. Standard arthroscopy equipment may be used. Initially,diagnostic arthroscopy may be performed to identify the appropriaterepair site. If a scaffold is used it should be compressible to allowintroduction through arthroscopic portals, incisions and equipment. Therepair material can be placed in the repair site by direct injection.After the procedure the arthroscopic portals can be closed and a steriledressing placed.

A subject includes, but is not limited to, any mammal, such as human,non-human primate, mouse, rat, dog, cat, horse or cow. In certainembodiments, a subject is a human.

The materials used in the invention are preferably biocompatible,pharmaceutically acceptable and sterile. As used herein, the term“biocompatible” refers to compositions (e.g. cells, tissues, matrices,etc.) that do not substantially disrupt the normal biological functionsof other compositions to which they contact. In selected embodiments,the present invention also contemplates biocompatible materials that areboth biodegradable and non-biodegradable.

As described above, each of the components of the repair material may beprepared sterilely. If however, one or more components is not retrievedor processed in a sterile manner then it can be sterilized prior toapplication to the subject. For instance the material (preferablywithout the cells) may be sterilized after production using gammairradiation, ethanol, autoclave sterilization or other knownsterilization methods.

As used herein, the term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the scaffold material or repair material. Theterm “physiologically acceptable” refers to a non-toxic material that iscompatible with a biological system such as a cell, cell culture,tissue, or organism. The characteristics of the carrier will depend onthe route of administration. Physiologically and pharmaceuticallyacceptable carriers include diluents, fillers, salts, buffers,stabilizers, solubilizers, and other materials which are well known inthe art. The term “carrier” denotes an organic or inorganic ingredient,natural or synthetic, with which the scaffold material is combined tofacilitate the application. The components of the pharmaceuticalcompositions also are capable of being co-mingled with the device of thepresent invention, and with each other, in a manner such that there isno interaction which would substantially impair the desiredpharmaceutical efficacy.

In some embodiments the repair material composition is injectable.Injectable compositions may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Pharmaceuticalformulations for injection may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the materials may also containsuitable stabilizers.

The collagen solution may be in the form of a liquid, gel or solid,prior to addition of the cells. Once the cells are added, the repairmaterial will begin to increase in gelation for application to the body.If the collagen solution is a liquid or gel the cells may be directlyadded to the solution.

Alternatively, the collagen solution may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. Neutralization agent may be added before or afterreconsistution. After the powder is reconsituted it is mixed with cellsto form the repair material.

As used herein, the term “gel” refers to the state of matter betweenliquid and solid. As such, a “gel” has some of the properties of aliquid (i.e., the shape is resilient and deformable) and some of theproperties of a solid (i.e., the shape is discrete enough to maintainthree dimensions on a two dimensional surface.). A gel may be providedin pharmaceutical acceptable carriers known to those skilled in the art,such as saline or phosphate buffered saline. Such carriers may routinelycontain pharmaceutically acceptable concentrations of salt, bufferingagents, preservatives, compatible carriers and optionally othertherapeutic agents.

An example of a gel is a hydrogel. A hydrogel is a substance that isformed when an organic polymer (natural or synthetic) is crosslinked viacovalent, ionic, or hydrogen bonds to create a three-dimensionalopen-lattice structure which entraps water molecules to form a gel. Apolymer may be crosslinked to form a hydrogel either before or afterimplantation into a subject. For instance, a hydrogel may be formed insitu, for example, at the repair site. In certain embodiments, therepair material forms a hydrogel within the repair site upon exposure tobody temperatures.

The repair material, including the collagen solution and the cells willbegin to set once it is created. The setting process can be delayed bymaintaining cold temperatures or it may be accelerated by warming themixture. In certain embodiments, a quick set composition of the repairmaterial is provided. The quick set composition is capable of forming aset scaffold within 10 minutes of mixture when the material is exposedto temperatures of greater than 30° C. In some embodiments formation ofthe scaffold takes approximately 5 minutes at such temperatures. Asdiscussed above, setting times can be further accelerated by optimizinginjection temperatures. The quick set composition is achieved bypreparing the collagen solution at concentrations and viscosities asdescribed herein. The quick set nature can be further enhanced by theaddition of non-toxic cross linking agents. Such compositions should beapplied quickly to the tissue defect to sufficiently set before closureof the defect and surgery area.

The invention also includes in some aspects kits for repair of rupturedor torn articular tissue. A kit may include one or more containershousing the components of the invention and/or for collecting or storingblood or cells and instructions for use. The kit may be designed tofacilitate use of the methods described herein by surgeons and can takemany forms. Each of the compositions of the kit, where applicable, maybe provided in liquid form (e.g., in solution), or in solid form, (e.g.,a dry powder). In certain cases, some of the compositions may beconstitutable or otherwise processable (e.g., to an active form), forexample, by the addition of a suitable solvent or other species (forexample, water or a cell culture medium), which may or may not beprovided with the kit. As used herein, “instructions” can define acomponent of instruction and/or promotion, and typically involve writteninstructions on or associated with packaging of the invention.Instructions also can include any oral or electronic instructionsprovided in any manner such that a user will clearly recognize that theinstructions are to be associated with the kit, for example, audiovisual(e.g., videotape, DVD, etc.), Internet, and/or web-based communications,etc. The written instructions may be in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which instructions can alsoreflects approval by the agency of manufacture, use or sale for humanadministration.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample (e.g., blood taken from a subject) andapplying to a subject. The kit may include a container housing collagen.The collagen may be in the form of a liquid, gel or solid (powder). Thecollagen may be prepared sterilely, packaged in syringe and shippedrefrigerated. Alternatively it may be housed in a vial or othercontainer for storage. A second container may have buffer solutionpremixed prepared sterilely or in the form of salts. Alternatively thekit may include collagen and some buffer premixed and shipped in asyringe, vial, tube, or other container. The mixture may or may notinclude neutralization agent. The neutralization agent may be includedin a separate container or may not be included in the kit.

The kit may have one or more or all of the components required to drawblood from a patient, process the sample into platelet concentrate orWBCs, and deliver the repair material to a surgical site. For instance,a kit for withdrawing blood from a patient may include one or more ofthe items required for such a procedure. For example, typically when aninjection is to be made, the patient's skin is cleansed with adisinfecting agent, such as an alcohol wipe; then a second disinfectingagent, such as iodine or Betadine may be applied to the skin; an area isusually isolated with a tourniquet to restrict the blood flow within theartery or vein making the vessel more visible before the needle isinserted, a needle attached to a collection device, such as a vacutainertube is injected through the patient's skin to withdraw the blood; theneedle is then removed and wiped clean; and the puncture site is coveredwith an absorbent pad until after hemostasis.

The accessories included may be specifically designed to allow thepractitioner to withdraw blood from the patient. For instance, theaccessories may include one or more of the following a tourniquet, askin penetration instrument, a device for housing blood, a collectiontube, disinfecting agents or post-injection bleeding patches.

The skin penetrating instrument for initiation of blood flow may be aconventional device such as a needle. The needle may be single or doubleended and may be of any gauge, preferably 21 or 23 gauge. It optionallyhas a safety sleeve, may be attached to a needle hub, and preferably isused with a conventional tube holder. The needle may also be part of aconventional syringe assembly including barrel and plunger. The needlemay be part of a conventional blood collection set in which apenetrating needle having a grasping means, such as wings, is connectedvia a hub and tubing to a delivery needle for puncture of a septum of anevacuated tube.

The device for housing the blood may be any type of container forreceiving the blood sample, such as, for example, a syringe barrel or itmay be a device to which the blood sample is transferred followingcollection, for example a tube. Preferred devices for housing the bloodare conventional tubes or vials having a closed end and an open end.Such tubes may have an internal volume of 100 μl to 100 ml. Devices tohouse the blood after it has been collected include for instance, vials,centrifuge tubes, vortex tubes or any other type of container. Thedevice for receiving the blood may be an evacuated tube in which theopen end is covered by a puncturable septum or stopper, such as avacutainer tube. Evacuated tubes are generally used with a conventionaltube holder and blood collection set for collection of multiple largerblood samples, and may contain any of a variety of conventional bloodanalysis additives, such as anticoagulants. Preferred anticoagulants arecitrate and ethylenediaminetetra acetic acid (EDTA).

The plasma, which contains the platelets, may be separated from thewhole blood. Any separation technique can be utilized, for example,sedimentation, centrifugation or filtration. Centrifugation can becarried out at about 500 g for about 20 minutes to obtain platelets. Thesupernatant, which contains the plasma, can be removed by standardtechniques. Filtration can be carried out by passing the whole bloodthrough a suitable filter that separates blood cells from plasma.

Optionally the kits may include disinfecting agents and post-injectionbleeding patches. A means for sterilizing the patient's skin in the areaof intended puncture, such as a disinfecting agent may be provided. Atypical and conventional disinfecting agent is a piece of fabriccommonly referred to as a gauze combined with a disinfectant. Sometypical disinfecting agents include rubbing alcohol, antibacterialagents, iodine, and Betadine, which may or may not be provided withapplication pads in individually sealed packets. The post-injectionbleeding patch can also vary from a relatively simple gauze pad plusadhesive strips, to a bandage.

When a blood draw is to be made, the practitioner may open the sealedkit; isolate a selected region of the patient's body, such as the lowerarm, with the tourniquet to restrict the blood flow within the regionand make the blood vessels more visible; clean the injection site withone or more of the sterilizing agents; attach the needle to thecollection tube; inject the needle into the patient's blood vessel andcollect the blood sample in the tube; withdraws the needle from theskin; and covering the puncture site with an absorbent pad. The bloodmay then be processed to produce a concentrate of platelets or whiteblood cells.

The kit may have a variety of forms, such as a blister pouch, a shrinkwrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, ora similar pouch or tray form, with the accessories loosely packed withinthe pouch, one or more tubes, containers, a box or a bag.

The kit may be sterilized after the accessories are added, therebyallowing the individual accessories in the container to be otherwiseunwrapped. The kits can be sterilized using any appropriatesterilization techniques, such as radiation sterilization, heatsterilization, or other sterilization methods known in the art.

The kit may also contain any other component needed for the intendedpurpose of the kit. Thus, other components may be a fabric, such asgauze, for removing the disinfecting agent after the sterilizing step orfor covering the puncture wound after the sample is drawn. Otheroptional components of the kit are disposable gloves, a support for thedevice for holding blood after the sample is taken, adhesive or otherdevice to maintain the fabric in place over the puncture wound.

The kit may include disposable components supplied sterile in disposablepackaging. The kit may also include other components, depending on thespecific application, for example, containers, cell media, salts,buffers, reagents, syringes, needles, etc.

The following examples are intended to illustrate certain aspects ofcertain embodiments of the present invention, but do not exemplify thefull scope of the invention.

EXAMPLES Example 1 Preparation of Collagen Solution and Testing ofProperties

A. Minimal Gelation Achieved with Some Formulations

1. Innocoll: Aliquots of Innocoll Collagen (Starting pH=4.1) were Made.

Weights of aliquots were 0.380 mg collagen, pH=4.1. One half of thesamples had 5 microliters NaHCO₃ added to bring pH to between 7.0 and8.0 and one half were not neutralized. 300 microliters fetal bovineserum was added to each aliquot. All solutions were monitored forgelation at 37° C. for 30 minutes. The solutions remained liquid, withviscosities similar to that of water (approximately 1 centipoise). Noincrease in viscosity with time was noted over the hour long period.

Identical experiments with Innocoll aliquots at starting pH=2.5 werealso performed. Additionally, experiments were performed using ratios ofcollagen:FBS of 1:1, 2:1 and 3:1. None of these materials produced agelled product at 37° C. The solutions remained liquid, with viscositiessimilar to that of water (approximately 1 centipoise). No increase inviscosity with time was noted over the hour long period.

2. EPC: Collagen Slurry was Obtained from Elastin Products Company(Owensville, Mo.), Product Number C857, Lot Number 698, Lyophilized TypeI Acid Soluble Collagen from Calf Skin.

Prepared according to the method of Gallop and Seifter Meth. Enzymol.,6, 635, (1963). In the method, fresh calf skin is extracted with 0.5 MNaOAc to remove non-collagen proteins. The soluble collagen is extractedwith 0.075 M sodium citrate pH 3.7 and precipitated as fibrils bydialysis against 0.02 M Na₂HPO₄. The product was soluble in 0.01 M to0.5 M acetic acid of maximum of 10 mg/ml and soluble in 0.075 M sodiumcitrate pH 3.7 and in dilute acetic acid 0.01 M to 0.5 M.

Before combining with the platelet component of the hydrogel, thecollagen slurry was mixed with 0.1M HEPES Buffer 1M solution (Cellgro,Mediatech, Inc, Herndon, Va.), 10× Ham's F-10 medium (MP Biomedicals,LCC, Aurora, Ohio), Antibiotic-Antimycotic solution (Cellgro, Mediatech,Inc., Herndon, Va.) and sterile water. The collagen slurry wasneutralized to a pH of 7.4 using 7.5% sodium bicarbonate (CambrexBioScience Walkersville, Inc., Walkersville, Md.). Mixtures ofcollagen-PRP were tested using ratios of collagen:FBS of 3:1. None ofthese materials produced a gelled product at 37° C. The solutionsremained liquid, with viscosities similar to that of water(approximately 1 centipoise). No increase in viscosity with time wasnoted over 1 hour or overnight.

3. SERVA: Collagen Slurry was Obtained from Serva, Product Number 47256,Lot Number 14902 Type I Rat Tail Collagen Solution at 4 mg/ml in 0.1%Acetic Acid (Heidelberg Germany.

The collagen slurry was mixed with 0.1M HEPES Buffer 1M solution(Cellgro, Mediatech, Inc, Herndon, Va.), 10× Ham's F-10 medium (MPBiomedicals, LCC, Aurora, Ohio), Antibiotic-Antimycotic solution(Cellgro, Mediatech, Inc., Herndon, Va.) and sterile water. The collagenslurry was neutralized to a pH of 7.4 using 7.5% sodium bicarbonate(Cambrex BioScience Walkersville, Inc., Walkersville, Md.).

Mixtures of collagen-PRP were tested using ratios of collagen:FBS of3:1. This material did not produce a gelled product at 37° C. Thesolutions remained liquid, with viscosities similar to that of water(approximately 1 centipoise). No increase in viscosity with time wasnoted over 1 hour or overnight.

4. VITROGEN: Collagen Slurry was Obtained from Cohesion Technologies(Palo Alto, Calif.). Vitrogen 100 Slurry, Lot Number C101636 with aCollagen Concentration of 3.1 Mg/Ml was Also Tested.

Vitrogen Collagen In Solution is 99.9% pure collagen as judged by SDSpolyacrylamide gel electrophoresis in conjunction with bacterialcollagenase sensitivity and silver staining techniques. The solution is95-98% Type I collagen with the remainder being comprised of Type IIIcollagen. Vitrogen Collagen In Solution is a native collagen as judgedby polarimetry and trypsin sensitivity, although it does contain a lowpercentage of nicked or shortened helices.

The collagen slurry was mixed with 0.1M HEPES Buffer 1M solution(Cellgro, Mediatech, Inc, Herndon, Va.), 10× Ham's F-10 medium (MPBiomedicals, LCC, Aurora, Ohio), Antibiotic-Antimycotic solution(Cellgro, Mediatech, Inc., Herndon, Va.) and sterile water. The collagenslurry was neutralized to a pH of 7.4 using 7.5% sodium bicarbonate(Cambrex BioScience Walkersville, Inc., Walkersville, Md.). Mixtures ofcollagen-PRP were tested using ratios of collagen:FBS of 3:1. Thismaterial did not produce a gelled product at 37° C. For the first thirtyminutes, the solutions remained liquid, with viscosities similar to thatof water (approximately 1 centipoise). No significant increase inviscosity with time was noted until one hour had passed.

5. Wake Forest Collagen Testing: Collagen Solutions were Made from PigSkin. The Skin was Washed with Water and the Hair and Subcutaneous FatRemoved. The Skin was Minced and Further De-Fatted with Acetone andWashed with Deionized Water.

The minced pieces were soaked in 10% NaCl at 4° C. for 24 hours thensoaked in a citrate buffer (pH 4.3) for 48 hr and homogenized in 0.5 Macetic acid at 4° C. The homogenate was then digested with pepsin at 4°C. for 24 hr and centrifuged. NaCl equivalent to 5% w/v was added tosalt-out atelo-collagen and the collagen washed with phosphate bufferand dissolved in 0.5 M acetic acid and dialyzed.

The aliquots of collagen were held on ice until they were mixed with FBSand Ham's F12. All solutions had a pH between 7.0 and 7.5. 150microliters of FBS and 50 microliters Hams F12 were added to each tube,mixed with vortexer, and placed in water bath at 37° C. One of fivealiquots gelled at 7 minutes. None of the other four gelled over the 30minute time period of the test. The tests were repeated for the 4collagen samples that did not gel. None of the repeated samples gelledduring the 30 minute time frame.

In order to confirm the accuracy of the gelation of the single samplethat produced a gelled product, a second trial was conducted to repeatthe study on the collagen type that did set in first trial. In thesecond trial no gelation was observed, even after 3 hours at 37° C. Theexperiment was attempted again with collagen and FBS only. The followingwas observed: ratio of 2:1 collagen:FBS produced no gelation and ratioof 3:1 collagen:FBS did gel when placed in water bath at 37° C. (gelsoftened over the remaining hour). Repeat testing of this collagen typeshowed inconsistent gelation which softened over time rather thancontinuing to hold shape.

B. Rapid Gelation Observed with Formulations of the Invention.

Pig Patellar Tendons were minced, placed into 10% NaCl solution toobtain salt-solubilized collagen. The collagen was homogenized andcentrifuged. The supernatant was aspirated and PRP was added. 5 of the 6samples tested resulted in rapid gelation when exposed to temperaturesof 37° C. In one sample no gelation was obtained.

Rat tail tendons were harvested. All steps were carried out usingsterile technique and solutions. Salt solubilized tendon fascicles werecentrifuged, the supernatant removed and replaced with acetic acid andenzyme to solubilized the collagen further. The resultant collagenslurry had a pH=3.0. Aliquots were made and neutralized with NaHCO₃ topH=7.0. 500 microliters of PRP were added to aliquots of 1 cc collagenslurry and vortexed (all on ice prior to mixing). The mixture was placedin a water bath at 37° C. Each of the samples formed a soft set gelwithin 5 minutes (partial gelation).

Additional aliquots with collagen slurry, and buffer containing F10culture media and antibiotics were tested. 150 microliters serum wasadded after neutralization with NaHCO₃ and NaOH. The collagen gel set in5 minutes at 37° C. on initial four tests. The fifth sample did not gel.Repeat testing showed most slurries made with this protocol would set,but not all (approx 60%).

The collagen solution was then prepared using different buffercomponents. With the addition of HEPES buffer to help maintain pHbetween 7.0 and 8.0 and adjusting other components of buffer(antibiotics, F10 and sterile water) to bring osmolarity to within 280to 350 mOsm/kg, we were able to get reliable gelation within 10 minutesfor over 90% of aliquots tested in trials.

C. Cell Viability in Collagen/Buffer Mixture:

Drop testing: One million pig primary outgrowth ACL cells weretrypsinized and added to a neutralized collagen slurry to produce adensity of cells of 1×105 cells/cc. FBS was added to produce a ratio of3:1 and 4:1 collagen:FBS. Drops were placed onto individual wells of atissue culture plate. The next day, some cell spreading was seen from 2of 3 gels. At day 4, cells were growing in 1 of 3 gels.

D. Evaluation of Whether the Proliferation of Pig ACL Cells Seeded inCollagen Gels is Affected by the Final Collagen Concentration of theGels.

The following methods were performed:

Targeted cells were seeded at 5×10⁵ cells/ml. The final collagenconcentrations in the gels was 3.4, 1.7 and 0.8 mg/ml. These finalconcentrations were calculated based on slurry #50's collagenconcentration (10.5 mg/ml), the fact that the slurry will be used atfull, ½ and ¼ strengths and how much the slurry is diluted when makingthe gels. Time points were taken at 1, 5 and 10 days, resulting in atotal of 9 data points (time/collagen concentration). Each data pointwas run in quadruplicate with 2 cell-free controls at each data point.

The total amount of cell-seeded gels was 36 ml. Enough gel for each datapoint was made using 1.5 ml slurry and 1.5 ml PRP. The PRP needed fordata points was 13.5 ml (1.5×9) and PRP needed for cell-free gels was2.2 ml. Total PRP needed was 15.7 ml.

The number of cells needed was 13.5×5×10^(5×3) (since PRP is diluted ˜3times when making the gel) which results in 202.5×10⁵ (or 20.3×10⁶)cells.

Day 1—Make Cell-Seeded Gels:

The cells were trypsinized, centrifuged, resuspended in complete mediaand counted to make sure there are enough cells for the assay. The cellsolution was centrifuged and the cells were resuspended in PRP such thatthe cell concentration in (at least) 13.5 ml PRP was 15×10⁵ cells/ml(PRP was diluted ˜3 times when making gels).

The gels were made using PRP with or without cells depending on thecase. 1 ml of gel was aliquoted on 42 wells of 12-well plates and placedin an incubator. After 1 hour the complete media was added on top ofgels and the cells were equilibrated in gels for ˜24 hours.

Days 2, 6 and 11—MTT Assay at Days 1, 5 and 10:

The plates were removed from the incubator and the media was aspirated.With a sterile spatula all gels were transferred into new 12-wellplates. 1 ml of MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye wasadded to each well of time point 1-day. The gels were incubated for 3hours. The MTT solution was aspirated off and discarded. 1 ml of sterile1×DPBS was added to each well. Plates were placed on a rotating platformand allowed gentle rinsing for 30 minutes. 200 μl aliquot was removedfrom each well and the absorbance was read for persistence of colorremoval. The remainder of DPBS was removed and the DPBS steps wererepeated twice. If absorbance levels from DPBS after 3^(rd) rinse arestill above 0.1 a 4^(th) rinse may be performed. Using a sterilespatula, the gels were detached from sides/bottom of wells andtransferred to new 3 ml tubes. 1 ml of detergent (20% SDS/Formamide) wasadded. The mixture was incubated for 5 hours. The tubes were removedfrom the incubator, briefly vortexed on high for—5 seconds and sampleswere spun down at 1500 rpm for 5 minutes. 200 μl of supernatant wastransferred to 96-well plate and the absorbance was read.

Results

Cell proliferation in vitro was seen in final collagen concentrations of0.8 to 3.4 mg/ml (starting collagen concentrations were 10.5, 5.3 and2.6 mg/ml). Cell number increased with time in culture for all groupsbetween 1 and 10 days. The results are shown in FIG. 10.

E. Viscosity of Collagen/Buffer Mixture:

At cold temperature viscosity was 70 cp and after heating to 37° C., theviscosity was assessed at 3200 cp at shear rate of 1/sec. At slowershear rate (0.3/sec), viscosity at 37° C. was determined to be6,000-11,000 cp.

F. Ability of Collagen/Buffer Solution to Stimulate Platelets to ReleaseGrowth Factors:

The collagen/buffer mixture (described in (B) above) was added to aplatelet mixture and the subsequent release of growth factors from theplatelets measured using an ELISA assay. The thrombin-free preparationswere compared to a preparation using bovine thrombin to stimulateplatelet release of growth factors. Similar growth factor release wasseen using the collagen slurry as a platelet activator as with thebovine thrombin as an activator. The results are described in Example 2.

G. Sterility

Multiple in vitro assays have been performed using the Collagen/Buffersolution described above in (B) with no evidence of bacterial or fungalcontamination or infection in any of the samples tested out to 10 daysin vitro and up to 9 weeks in vivo.

H. In Vivo Testing of Collagen/Buffer Solution

Model: Pig total ACL transection was treated with collagen slurry/buffermixed with animal's own PRP in the operating room. The mixture wasinjected into the gap between the cut ligament ends. Adding theCollagen/Buffer solution to the autologous PRP resulted in more thandoubling of the yield strength of the healing ligament after four weeksin vivo compared with the use of sutures alone for the repair. FIG. 9 isa graph depicting results of the in vivo pig total ACL transectiontreated with collagen slurry/buffer.

Example 2

In this Example, type I collagen was used to stimulate activation of thefibrin clotting mechanism and platelet activation. Initially, we testedcollagen to determine if it would result in more sustained release oftwo growth factors used as markers of platelet function, namely TGF-β1and PDGF-αβ, when compared with the use of exogenous thrombin. Secondly,we tested whether the amount of this release would be dependent on theplatelet concentration in the PRP. The release profiles of these growthfactors from three types of collagen-PRP gels were compared with therelease profile from a PRP clot created with exogenous bovine thrombinover 10 days. Additionally, we determined whether the growth factorrelease from collagen-activated PRP hydrogels would cause a physiologicchanges in ACL cells in terms of 1) cellular metabolism of growthfactors, 2) cellular proliferation within the gels and 3) cell-mediatedgel contraction.

Materials and Methods Preparation of Platelet-rich Plasma:Centrifugation Method

Three hundred milliliters of whole blood was drawn from each of fivehematologically normal volunteers meeting all criterion of the AmericanAssociation of Blood Banks (Food and Drug Administration, Center forBiologics Evaluation and Research). Blood was collected in a bag tocontain 10% acid-citrate dextrose at the Center for Blood Research(Boston, Mass.). Forty five ml of whole blood from each patient wascentrifuged for 6 minutes at 200 g (Beckman GS-6 Centrifuge, Fullerton,Calif.). The supernatant was aspirated and collected as PRP. Twoadditional groups of PRP samples were made using the Harvest Smart PreP2System (Harvest Technologies, Plymouth, Mass.) as noted below.

PRP Preparation Using the Smart PreP2 System: Platelet ConcentrateMethod

PRP was also produced using the Harvest Smart PreP2 System (HarvestTechnologies, Plymouth, Mass.). PRP was produced by the methodrecommended by the manufacturer. Fifty four cc of whole blood wasanticoagulated using 10% acid-citrate dextrose and transferred to theblood chamber of the device, and 2 ml ACD was placed in the plasmachamber of the disposable blood processor (DP). The blood is centrifugedin a container with a floating shelf designed to rise to just below thebuffy coat/red blood cell interface. Following the separation of plasmafrom the red blood cells, the centrifuge slows, and the platelets,plasma and white blood cells are decanted into the plasma chamber. Whenthe plasma decant is complete, a second centrifugation step is used toform a pellet of platelet concentrate in the bottom of the plasmachamber. The plasma chamber contains the platelet concentrate (abutton-like precipitate) and platelet poor plasma (supernatant). Thecomplete process is entirely automatic and completed in approximately 14minutes. Approximately ⅔ of the platelet poor plasma (PPP) is removed.The platelet concentrate (PC) is then resuspended in the remaining PPP.

PRP Preparation Using the Smart PreP2 System: RBC-Reduced Method

PRP in this group was prepared using the platelet concentrate as abovewith an additional step to remove the majority of erythrocytes in thePRP. To accomplish this, 30 ml of platelet concentrate from each patientwas centrifuged in the Smart PreP2 system for an additional 2 minutes.The supernatant is then aspirated and kept as the RBC-reduced (RBC-red)PRP.

Samples of whole blood and platelet rich plasma preparations wereanalyzed for complete blood count with differential to determine initialand final platelet and white blood cell concentrations (Table 1 andTable 2)

TABLE 1 Platelet counts for each PRP preparations. Baseline CentrifugedPC RBC Reduced Patient # Plt Count (% of baseline) (% of baseline) (% ofbaseline) 1 316 434 (137%) 919 (291%) 1287 (407%) 2 204 286 (140%) 860(422%) 1000 (490%) 3 194 249 (128%) 715 (368%)  794 (409%) 4 318 573(180%) 1215 (382%) 1385 (436%) 5 246 479 (195%) 1057 (430%) 1131 (460%)Avg 256 404 (158%) 953 (373%) 1119 (438%)

TABLE 2 Differential in total cells/microliter. Centri- Patient Baselinefuged PC RBC Reduced # WBC GRN WBC GRN WBC GRN WBC GRN 1 3,500 2,0001,400 100 7,700  700 7,600 300 2 3,900 2,600 1,000 0 7,600 1,000 5,400200 3 6,700 4,800   500 0 13,600 5,600 4,900 600 4 5,500 3,300 1,100 10016,000 4,300 8,000 1,300 5 5,000 3,300   500 0 11,000 3,300 6,100 700Avg 4,920 3,200   900 40 11,180 2,980 6,400 620

Manufacture of Acid-Soluble Collagen Used in the Hydrogels

Rat tails were obtained from control breeder rats undergoing euthanasiafor other Institutional Animal Care and Use Committee approved studies.The rat-tail tendons were sterilely harvested, minced, and solubilizedin an acidified pepsin solution to obtain the acid soluble collagen.Collagen content within the slurry was adjusted to greater than 5 mg/mlusing 0.01N hydrochloric acid. Before combining with the plateletcomponent of the hydrogel, the collagen slurry was mixed with 0.1M HEPESBuffer 1M solution (Cellgro, Mediatech, Inc, Herndon, Va.), 10× Ham'sF-10 medium (MP Biomedicals, LCC, Aurora, Ohio), Antibiotic-Antimycoticsolution (Cellgro, Mediatech, Inc., Herndon, Va.) and sterile water. Thecollagen slurry was neutralized to a pH of 7.4 using 7.5% sodiumbicarbonate (Cambrex BioScience Walkersville, Inc., Walkersville, Md.).

Platelet Activation: Exogenous Thrombin Group

Five milliliters of calcium chloride (100 mg/ml) were added to 5,000 IUbovine thrombin (Bovine Thrombin—JMI, Jones Pharma Inc, Bristol, Va.) toproduce a 1,000 IU/ml solution. 80 ml of the thrombin solution was thenadded to 720 ml of the Platelet Concentrate group for each patient.Duplicate samples of the mixture were injected into 2 ml centrifugetubes and allowed to form a clot. Clots were weighed and placed in a 37°C. incubator for 20 minutes prior to transfer to sterile 12-well plates.One milliliter of Dulbecco's Modified Eagle's Medium (DMEM, Cat#10013CV,Cellgro, Mediatech, Inc., Herndon, Va.) with 2% antibiotics (Cellgro,Mediatech, Inc., Herndon, Va.) was added to each clot. Samples werecultured in a 37° C. humidified incubator.

Platelet Activation: Collagen Groups

For each sample, an equal volume of PRP and collagen hydrogel were mixedand heated to 30° C. over 1 minute. Duplicate samples of eachcollagen-PRP mixture were injected into two 2 ml centrifuge tubes. Thiswas repeated for all test groups. Gels were weighed and placed in a 37°C. incubator for 20 minutes prior to transfer to sterile 12-well plates.One mL of DMEM with 2% antibiotics (Cellgro, Mediatech, Inc., Herndon,Va.) was added to each gel. Samples were cultured in a 37° C. humidifiedincubator.

Additionally, a collagen hydrogel-only (no PRP) was also made and therelease evaluated at 12 hours, 1 day, 3 days and 5 days.

Measurement of Growth Factor Levels:

At each time point (12 hours, 1, 3, 5, 7 and 10 days) media wasaspirated from around each sample and replaced with 1 mL of fresh media(serum-free DMEM with 2% antibiotics added). Media samples were storedin cryovials in a −80° C. freezer until all samples were collected.Concentrations of human PDGF αβ, TGF β₁ and VEGF were determined usingthe commercially available Quantikine colorimetric sandwich ELISA kits(R&D Systems, Minneapolis, Minn.). Assays were performed in duplicate onmedia samples as described in the instructions of the manufacturer.Dilutions of 1:20 (12 hour samples) and 1:10 (day 1, day 3, day 5, day 7and day 10 samples) were used for samples in the PDGFαβ assay; adilution of 1:10 was used for all samples in the TGF β₁ assay; and nodilution was used for the VEGF assay. These dilutions were accounted forin analysis.

The media concentration of each growth factor was determined using theELISA kit after performing the dilutions described above. The plasmatotal TGF β₁ was assayed after acid activation of the plasma by adding20 microliters of 1N HCl to 40 microliters of media sample. The reactionsolution was mixed and incubated at room temperature for 10 minutesbefore it was neutralized by with microliters of 1.2N of NaOH/0.5 MHEPES. It was further diluted to 1:10 in calibrator diluent before itwas added to the ELISA plate.

For each growth factor, the standard curve was produced by a 2-foldserial dilution of a known concentration of growth factor provided inthe kit to make final concentrations of 0, 31.2, 62.5, 125, 250, 500,1000 and 2000 pg/ml. The color change of the final reaction was measuredat a wavelength of 450 nm for the optical density and the standard curveconcentrations vs absorbances was linear using a four parameter logisticfit curve. The reported minimal detection limit of TGF-β1 was 4.61pg/ml, 9.0 pg/ml for VEGF and 1.7 pg/ml for PDGF-αβ.

Due to the media sampling technique described above, growth factorconcentrations reported in the results section reflect the growth factorrelease in the time period since the prior media change. For 12 hoursand day 1, this is a 12 hour release and for day 3, day 5 and day 7, itis a 48 hour release.

Analysis: The cumulative TGFb release was measured after 1, 5 and 10days of culture of gels seeded with 3×105 ACL cells. The effect of theseeded cells on TGFb release was calculated by subtracting thecumulative TGFb release from cell-free gels at each time point from thecumulative TGFb release from the cell seeded gels at the same time point(both values calculated per ml gel to account for possible differencesin gel sizes during gel manufacture).

The cumulative PDGF release was measured after 1, 5 and 10 days ofculture of gels seeded with 3×105 ACL cells. The effect of the seededcells on PDGF release was calculated by subtracting the cumulative PDGFrelease from cell-free gels at each time point from the cumulative PDGFrelease from the cell seeded gels at the same time point (both valuescalculated per ml gel to account for possible differences in gel sizesduring gel manufacture).

The cumulative VEGF release was measured after 1, 5 and 10 days ofculture of gels seeded with 3×105 ACL cells. The effect of the seededcells on VEGF release was calculated by subtracting the cumulative VEGFrelease from cell-free gels at each time point from the cumulative PDGFrelease from the cell seeded gels at the same time point (both valuescalculated per ml gel to account for possible differences in gel sizesduring gel manufacture).

Effect on Cell Proliferation: MTT Assay

Collagen-PRP hydrogels and thrombin-PRP clots containing 3×105 cells/mlwere prepared as follows. Primary outgrowth human ACL cells werecultured from explants obtained from 2 women (ages 15 and 22) undergoingACL reconstruction. Explants were cultured in media containing 10% FBS(HyClone Inc., Cat. #16777-006, South Logan, Utah), 1% AB/AM (MediaTech, Inc., Cat. #30004067, Herndon, Va.) and media changed 2 times perweek until confluent cultures established. The cells were trypsinizedand replated once onto T-75 flasks until use. First passage ACL cellswere trypsinized, resuspended in complete media, counted and fourpellets containing 6×10⁶ cells were prepared in 15 cc centrifuge tubes.Each pellet was resuspended in 6.5 cc of one of four solutions: PRPprepared by centrifugation, PC PRP, RBC-reduced PRP or normal phosphatebuffered saline (EMD Chemicals, Cat. #: B10241-34, Gibbstown, N.J.).

Cell proliferation was measured using the MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay;this assay measures the ability of a cell's mitochondrial dehydrogenaseenzymes to convert yellow, soluble MTT salt into purple formazan salt.The MTT was prepared at a concentration of 1 mg/ml in serum-free DMEMfrom the sterile stock MTT solution (5 mg/ml PBS). After the media wasremoved from each well, a sterile spatula was used to transfer each gelto a sterile 12-well plate; this allowed only those cells proliferatingin the gel to be labeled by the MTT. 1.2 ml of MTT solution (1 mg/ml)was added to each well. Each gel was fully immersed in the MTT solution.After the MTT was added, the plates were incubated for 3 hours (37 C, 5%CO2). Subsequently, the excess MTT solution was removed and 1 ml ofsterile 1×PBS was added to each well, placed on a vertical agitator(Fisher Scientific Clinical Rotator, 100 rpm) and left to rinse at roomtemperature for 30 minutes. Afterwards, 150 microliters of PBS wasremoved from each well, transferred to a sterile 96-well plate and theabsorbancies read at 562 nm. This rinse was repeated until all PBSaliquots read absorbencies under 0.100 nm. All PBS was then removed andeach gel transferred with a sterile spatula into a sterile 3.0 mlcentrifuge tube. The gels and ? formazan crystals were then dissolved byadding 1 ml of a detergent containing 20% aqueous SDS/formamide (1:1volume ratio) to each tube and incubating for 5 hours in a 37 C waterbath. Finally, the tubes were centrifuged for 5 minutes at 1500 rpm, andaliquots of the supernatant from each tube (200 microliters) were thentransferred onto a sterile 96-well plate. The absorbencies were measuredat 562 nm, and the cell concentrations determined.

We were unable to assess the effect of the PC on cell proliferation asthe control readings for the cell free gels were higher than theabsorbance reader tolerance, likely due to the high number of red bloodcells in this PRP preparation.

MTT controls were prepared identically to the method above differingsolely in their absence of cells. MTT protocol was again performed at 1,5, and 10 days allowing the controls to be determined and when appliedto the MTT results from the cell seeded hydrogels, the effect of eachgels' cells isolated and compared.

Collagen Gel Contraction Assay

Both fibroblast and PRP-mediated collagen gel contraction was assessed.The degree of contraction of collagen gels was determined by measuringthe area of each gel over time in culture. Every two days (day 1, day 3,day 5 and day 7), the length and width at the gel midpoint was measuredusing a millimeter ruler and recorded. Comparisons betweenfibroblast-seeded gels with and without PRP and cell-free gels with andwithout PRP were made.

Statistical Analysis

Two-factor ANOVA for group and time was used to compare the growthfactor release of the collagen-PRP hydrogels with those of thethrombin-PRP clots, with values of p<0.05 considered statisticallysignificant. Bonferroni-Dunn post hoc testing was used to determine thesignificance of observed differences between groups in a pairwiseanalysis.

Results

Hypothesis One: That the Use of Collagen as a Platelet Activator wouldResult in a More Sustained Release of Growth Factors from a PRP Gel

In both the bovine thrombin-activated and collagen-activated PRP gels,the highest release of PDGF-αβ and TGF-β1 occurred in the first twelvehours (FIGS. 1 and 2). For time points greater than 3 days (delayedrelease), there was no difference in release of PDGF-αβ or TGF-β1between the bovine thrombin-activated and collagen-activated groups. Inboth groups, the release of PDGF-αβ at 10 days, was higher than 1.9ng/ml and for TGF-β1, the release at 10 days was higher than 15 ng/mlfor both bovine-activated and collagen-activated PRP gels.

The results are shown in FIGS. 1 and 2. FIG. 1 is a graph depicting therelease of PDGF-αβ over time from bovine thrombin-activated (BT) andcollagen-activated (Centr, PC and RBC Reduced) PRP hydrogels. Therelease of TGF-β1 over time from bovine thrombin-activated (BT) andcollagen-activated (Centr, PC and RBC Reduced) PRP hydrogels is shown inFIG. 2.

Hypothesis Two: That Platelet Number in the PRP would Affect the Releaseof Growth Factors from the PRP Gels.

There was a strong positive correlation between platelet count in thePRP preparation and TGF-β1 and PDGF-αβ release. For TGF-b, this wasstrongest at the 12 hour time point (r2=0.608) and remained positive atthe 10 and 12 day time points (r2>0.35 for both correlations). Apositive correlation was also found between platelet concentration inthe gel and PDGF release, particularly at the 12 hour time point(r2>0.35). The results are shown in FIGS. 3 and 4.

TGF-β1 release as a function of platelet concentration in the PRP at 12hours after platelet activation is depicted in FIG. 3. FIG. 4 shows thePDGF-αβ release from the PRP gels as a function of plateletconcentration in the PRP at 12 hours after platelet activation.

There was a strong positive correlation between platelet concentrationin the gels and gel contraction at all time points (r2>0.64 at all timepoints), suggesting the contraction of the gels was platelet-mediated.Much lower correlations were found between granulocyte counts and gelcontraction (r2<0.30 for all correlations) and was more likely due tothe correspondence between platelet and granulocyte content in the PRP(r2=0.35).

Hypothesis Three: That the Growth Factor Release from Collagen-ActivatedPRP Hydrogels would Cause a Physiologic Changes in ACL Cells in Termsof 1) Cellular Metabolism of Growth Factors, 2) Cellular Proliferationwithin the Gels and 3) Cell-Mediated Gel Contraction.

1) Cellular Metabolism of Growth Factors in Collagen-PRP Gels

Less TGF-β1 and PDGF-αβ eluted from the cell-seeded gels than from thecell-free gels, suggesting the cells were metabolizing the TGF-GlandPDGF-αβ. There was no significant difference among groups (two-factorANOVA with p>0.2 for group and p>0.4 for time). In contrast, more VEGFeluted from the cell-seeded gels, suggesting the cells were producingadditional VEGF. There was no significant difference between the groups,however VEGF release did increase over time in culture (two factor ANOVAwith p>0.15 for group and p<0.02 for time, BFD post-hoc testing p<0.012for comparison between 1 and 10 day values). On average, more than 4times as much VEGF was eluted from the cell-seeded gels than from thecell-free gels.

The PDGF-αβ elution over time from the cell-seeded PRP hydrogels isshown in FIG. 5. The negative values over time suggest cell-basedconsumption of the PDGF-αβ. VEGF elution over time from the cell-seededPRP hydrogels is shown in FIG. 6. The positive trend over time suggestscontinuing greater production than consumption of the VEGF by the ACLcells.

2) Cellular Proliferation within the Gels

The incorporation of centrifuged or RBC-depleted platelet rich plasma inthe collagen hydrogel resulted in a significant increase in cell numberwithin the gels over 10 days of culture in vitro (one factor ANOVA,p<0.009) with an almost two-fold increase in cell number between saline(0.165+/−0.03 mean+/−sd) and centrifuged (0.316+/−0.04) groups and amore than 2-fold difference between the saline and RBC-depleted(−0.359+/−0.11) groups. No significant difference was seen between thecentrifuged and RBC-D groups (BFD, p>0.40). The results are shown inFIG. 7.

3) ACL Cell-Mediated Gel Contraction

The addition of ACL cells to the gels resulted in a stabilization of gelsize during the 10 days of culture (two factor ANOVA, p<0.006 for timeand p<0.001 for group, BFD p>0.003 for all comparisons between timepoints except between days 0 and 1 where p<0.001). The results are shownin FIG. 8.

Effect of Growth Factor Consumption on Gel Contraction

There was a positive correlation between gel contraction and TGF-β1consumption, that is, the more TGF-β1 consumed by the cells, the greaterthe cell-mediated contraction of the gels. This was most significant inthe RBC reduced group (r2=0.38) and less so in the PC (r2=0.24) andCentrifuged (r2=0.11) groups. There was also a positive correlationbetween gel contraction and PDGF consumption, that is, the more PDGFconsumed by the cells, the greater the cell-mediated contraction of thegels. This was most significant in the Centrifuged (r2=0.46) and PCgroups (r2=0.42) than in the RBC red group (r2=0.27). In contrast, themore VEGF produced by the cells, the greater the cell-mediatedcontraction of the gels (r2=0.38). This was only significant in theCentrifuged group (r2=0.38) and was not seen in the PC (r2=0.11) or theRBC reduced group (r2=0.004).

Example 3

The components of a preferred collagen solution of the invention wastested to identify components.

Methods

Collagen from various sources (Cellagen, MP Biomedicals, Solon, Ohio(shown as lane 4 in FIG. 11); Elastin Products Company, Inc.,Owensville, Mo. (shown as lane 5 in FIG. 11); StemCell Technologies(shown as lane 6 in FIG. 11), Becton Dickinson, Franklin Lakes, N.J.(shown as lane 7 in FIG. 11); fluorescein-labeled collagen (shown aslane 8 in FIG. 11)) were prepared into aliquot samples. Total proteincontent of each collagen sample was determined using a colorimetricassay (BCA Protein Assay Kit, Rockford, Ill.) in order to aliquotsamples containing equal protein content. The aliquots were treated withSDS and (3-mercaptoethanol and placed at 100° C. for five minutes fordenaturation and then loaded onto a 4-12% SDS-PAGE gel. The gels werestained with Coomassie Blue (Bio-Rad, Hercules, Calif.) and washed witha 7.5% acetic acid and 5% methanol destaining solution.

Results

The results of the staining of the SDS-PAGE gel are shown in FIG. 11.The results demonstrated that the commercially available productscontained relatively pure Type I collagen (Lanes 4-8). Lanes 1-2 serveas negative controls. The material shown in Lane 3 of the gel in FIG. 11was prepared according to the methods described herein, i.e. Example 1,“manufacture of acid soluble collagen used in the hydrogels”. Thecollagen preparation of the invention produced additional bandsmigrating at about 39KD, signifying additional proteins present in thispreparation. Two additional bands were seen at approximately 39KD,consistent with decorin and biglycan, as well as several other bands.These bands were only observed in the collagen preparations preparedaccording to the methods of the invention, and not in any of thecommercially available collagen products tested.

Example 4

A challenge for stimulation of ACL healing has been to create anactivated delivery system that provides for the release of growthfactors found during the successful wound healing process in other softconnective tissues The addition of white blood cells (WBCs) to collagensolutions was tested. Surprisingly it was discovered that detectableconcentrations of WBCs in the platelet rich plasma-collagen solutionsdescribed herein had a significant effect on the immediate release ofVEGF from the hydrogels.

Methods

Methods for Preparation of Platelet-rich Plasma, PRP preparation usingthe Smart PreP2 system, Platelet and RBC-Reduced Method, Manufacture ofacid-soluble collagen used in the hydrogels, and Platelet Activation:Collagen Groups were performed as described in Example 2.

Samples of whole blood and platelet rich plasma preparations wereanalyzed for complete blood count with differential to determine initialand final platelet and white blood cell concentrations. The data isshown in Table 2 above.

Measurement of Growth Factor Levels:

VEGF release from each gel was measured at 12 hours. Media was aspiratedfrom around each sample and replaced with 1 ml of fresh media(serum-free DMEM with 2% antibiotics). Media samples were stored in 1.3ml cryovials in a −80° C. freezer until all samples were collected.Concentrations of human VEGF were determined using the commerciallyavailable Quantikine colorimetric sandwich ELISA kits (R&D Systems,Minneapolis, Minn.). Assays were performed in duplicate on media samplesas described in the instructions of the manufacturer. No dilution wasused for the VEGF assay.

For each growth factor, the standard curve was produced by a 2-foldserial dilution of a known concentration of growth factor provided inthe kit to make final concentrations of 0, 31.2, 62.5, 125, 250, 500,1000 and 2000 pg/ml. The color change of the final reaction was measuredat a wavelength of 450 nm for the optical density and the standard curveconcentrations vs absorbances was linear using a four parameter logisticfit curve. The reported minimal detection limit of TGF-β1 was 4.61pg/ml, 9.0 pg/ml for VEGF and 1.7 pg/ml for PDGF-αβ.

Results

The results of the study are shown in FIG. 12. Linear regressionanalysis demonstrated a positive correlation between WBC, and inparticular granulocyte, count and VEGF release at the 12 hour timepoint, with r²=0.35. The results demonstrate that the inclusion of WBCsin the collagen-PRP materials of the invention can result in improvedconditions for healing and tissue repair.

Example 5 Injection Temperature Significantly Effects In Vitro and InVivo Performance of Collagen-PRP Hydrogels

We have demonstrated the efficacy of the use of collagen-PRP hydrogelsto stimulate healing of the anterior cruciate ligament (ACL) afterpartial and complete transection in animal models. These hydrogels arethought to serve as a substitute provisional scaffold in the ACL woundsite. Important rheologic properties of the provisional scaffold includeits gelation characteristics (including final modulus and time togelation). As described above, the modulus of the provisional scaffoldmust be sufficient to maintain the provisional scaffold analog in thewound site and to allow it to deform in a similar fashion to thesurrounding wound edges. A hydrogel with a modulus that is too low ismore likely to flow out of the wound site before wound healing can bestimulated. The time to gelation is also important, as in a surgicalprocedure, a provisional scaffold substitute that can achieve gelationin five minutes is far more practical than a hydrogel that requires 60or more minutes to become firm enough to allow for closure of theoperative site.

In this experiment, we tested the mechanical perturbation during theaccumulation of collagen crosslinks as well as the temperature at whichthis final perturbation occurred and demonstrated that they had asignificant effect on the mechanical properties of the provisionalscaffolds in vitro, and also in turn significantly effected the functionof these materials in an in vivo model of ACL repair.

Materials and Methods In Vitro Study: Experimental Design

Acid soluble collagen was neutralized and combined with platelet richplasma to form aliquots of provisional scaffold matrix. Each aliquot wasthen mixed under specific heating and mixing parameters using anautomated device which could accurately control the heating voltage,mixing speed and mixing time while simultaneously recording temperaturewithin the gel. After processing, aliquots were injected onto the plateof a small oscillation rheometer and modulus and time to gelationrecorded.

Preparation of Platelet-Rich Plasma (PRP)

A total of one thousand two milliliters of whole blood was drawn fromtwo hematologically normal pigs undergoing other Institutional AnimalCare and Use Committee approved studies. Blood was collected in a bagcontaining 10% by volume acid-citrate dextrose. The blood wastransferred into fifteen milliliter centrifuge tubes, ten millilitersper tube. The tubes were centrifuged for six minutes at 150 g's (GH 3.8rotor, Beckman GS-6 Centrifuge, Fullerton Calif.). The supernatant wascollected as PRP, and complete blood counts (CBC's) were taken.

Manufacturing of Acid-Soluble Collagen Used in Hydrogels

The collagen used in this study was derived from rat tails which wereobtained from control breeder rats undergoing euthanasia for otherInstitutional Animal Care and Use Committee approved studies. Therat-tail tendons were sterilely harvested, minced, and solubilized. Thecollagen content in the resulting slurry was found to be >5 mg/ml. Thesame collagen slurry was used in all experiments.

The collagen slurry was neutralized using HEPES Buffer (Cellgro,Mediatech, Inc, Herndon, Va.), Ham's F-10 medium (MP Biomedicals, LCC,Aurora, Ohio), Antibiotic-Antimycotic solution (Cellgro, Mediatech,Inc., Herndon, Va.) and sterile water. 7.5% sodium bicarbonate (CambrexBioScience Walkersville, Inc., Walkersville, Md.) was used to neutralizethe acidic slurry to a pH of 7.4. Aliquots of provisional scaffoldanalogs were created by combining equal amounts of PRP and theneutralized collagen and kept on ice until mixing as outlined below.

Apparatus for Mixing and Heating the Gels

Mixing speed, mixing time, and heating rate were controlled using acradle designed and built by TNCO Inc. (Whitman, Mass.). An auger wasdesigned to fit inside the 6 cc syringe held in the cradle. This allowedfor mixing of the collagen hydrogel components. This device had a motorwhich was coupled to the auger to allow for control of mixing speed andtime, and a heating pad under the syringe that allowed for control ofheating rate. The cradle was connected to a notebook computer running acustom LabView (Austin, Tex.) application which allowed for control ofthe variables, and logging of feedback data.

The experiments performed tested three different mixing speeds (50 RPM,100 RPM, and 150 RPM), three different mixing times (30 seconds, 60seconds, and 120 seconds), and three different heating rates (9 mV, 11mV, and 13 mV). All combinations of those parameters were tested as seenin Table 3. The final temperature of the gels were recorded for thesemixing conditions. Additional triplicate gels having an injectiontemperature of 24° C.-26° C., 26° C.-28° C., 28° C.-30° C., and 30°C.-32° C. were also tested. The additional gels were prepared by mixingat 100 RPM and heating at 11 mV for the time necessary for the gel toreach the required final temperature.

TABLE 3 Gel Preparation Parameters Mixing Speed Mixing Time Heating RateRPMs (sec) (mV) 50 60 11 100 60 11 200 60 11 50 30 11 50 120 11 50 60 950 60 13

Preparation of Gels

One milliliter aliquots of the acid soluble collagen were measured into5 ml cryotubes. An appropriate quantity of buffer was added to the tubeand vortexed for five seconds. The auger was then placed into thesyringe, and used to aspirate the neutralized collagen. An equal amountof PRP as acid soluble collagen was then aspirated into the samesyringe. The syringe was affixed in the cradle and mixed accordingly.

Mechanical Testing

Mechanical properties of the gels were determined using Cone on PlateSmall Amplitude Oscillatory Shear Rheometry using a TA Instruments AR1000 Rheometer (New Castle, Del.). The rheometer was fitted with a 60 mm1° acrylic cone, and the base plate was heated to 25° C. A gel wasprepared as described above, and one milliliter of the collagen-PRP gelwas dispensed onto the rheometer plate. The cone was lowered so that thegel was situated in a 38 μm layer between the cone and plate, andsubjected to a 1% oscillatory strain.

The viscoelastic complex modulus of the gel was recorded as the gelationprogressed. Elastic modulus (G′), inelastic modulus (G″), and phaseangle were measured for all of the gels.

In Vivo Studies: Experimental Design

Five 30 kg female Yorkshire pigs were used in the study. Four animalshad bilateral ACL transactions and for each of these, one side wastreated with a suture repair augmented with a collagen-PRP hydrogel,while on the contralateral side, the transection was treated with suturerepair without hydrogel. In the remaining animal, unilateral surgery wasperformed with the augmented repair and the contralateral side left as acontemporary intact control. One of the animals developed apost-operative seroma which was treated with antibiotics on thecollagen-PRP side. This knee was excluded from the study. Therefore,there were a total of four knees in the augmented repair group and fourknees in the non-augmented group. All animals were survived to 14 weeksand then underwent MRI evaluation and euthanasia. Knees were immediatelyharvested and frozen until biomechanical testing. Load to yield, load tofailure, maximum stiffness and displacement to failure were measured.

Surgical Procedure

Institutional Animal Care and Use Committee approvals were obtained forthis study prior to any surgical procedures. Five 30 kg female Yorkshirepigs were used in this study. The pigs were pre-medicated with telazol4.4-6.6 mg/kg IM, xylazine 1.1-2.2 mg/kg IM, and atropine 0.04 mg/kg.They were intubated and placed on isoflurane 1-3% for anesthesiamaintenance. After anesthesia had been obtained, the pigs were weighedand placed in the supine position on the operating room table. Both hindlimbs were shaved, prepared with chlorhexidine followed by betadynepaint and sterilely draped. No tourniquet was used. To expose the ACL, afour-centimeter incision was made over the medial border of the patellartendon. The incision was carried down sharply through the synovium usingelectrocautery. The fat pad was released from its proximal attachmentand partially resected to expose the intermeniscal ligament. Theintermeniscal ligament was released to expose the tibial insertion ofthe ACL. A Lachman maneuver was performed prior to releasing the ACL toverify knee stability. Two #1 Vicryl sutures were secured in the distalACL stump using a modified Kessler stitch. The ACL was transectedcompletely at the junction of the middle and proximal thirds using a No12 blade. Complete transection was verified visually and with a repeatLachman maneuver that became positive in all knees with no significantendpoint detected after complete transection. All knees were irrigatedwith sterile saline to remove synovial fluid before suture anchorplacement. An absorbable suture anchor (TwinFix AB 5.0 Suture Anchorwith DuraBraid Suture (USP#2); Smith and Nephew, Inc, Andover Mass.) wasplaced at the back of the femoral notch. The knee was irrigated with 500cc of sterile normal saline to remove all synovial fluid. Oncehemostasis had been achieved, a collagen sponge was soaked in coldcollagen-PRP hydrogel and threaded onto sutures and up into the regionof the proximal ACL stump in the notch. The sutures were tied usingmaximum manual tension with the knees in resting flexion (approximately70°-40° short of full extension in these animals). A second batch ofcollagen-PRP hydrogel was mixed by sequentially drawing up equalaliquots of neutralized collagen solution and autologous PRP into themixing and heating device and mixing for 1 minute at 50 rpm and 13 mVwhich resulted in injection temperatures between 28.9 and 32.4° C. Thismixture was then placed over the ACL repair to fill the intercondylarnotch. The knee was left in resting extension while the identicaltechnique of suture anchor repair was performed with an identicalcollagen sponge, but without the addition of the collagen-PRP hydrogel.The incisions were closed in multiple layers with absorbable sutures.

The animals were not restrained post-operatively, and were allowed adlib activity. Once the animals recovered from anesthesia, they werepermitted to resume normal cage activity and nutrition ad lib. Buprenex0.01 mg/kg IM once and a Fentanyl patch 1-4 ug/kg transdermal wereprovided for post-operative analgesia. All animals were weight bearingon their hind limbs by 24 hours after surgery. After fourteen weeks invivo, the animals were again anesthetized and underwent in vivo MRimaging using the protocol detailed below.

After the magnetic resonance images had been obtained, the animals wereeuthanized using Fatal Plus at 1 cc/10 lbs. No animals had any surgicalcomplications of difficulty walking normally, redness, warmth andswelling of the knee, fever or other signs of infection that would havenecessitated early euthanasia.

The six intact control knees were obtained from age-gender- andweight-matched animals after euthanasia following surgical procedures tothe chest. The hind limbs were frozen at −20° C. for three months andthawed overnight at 4° C. before mechanical testing. All other testingconditions for these knees were identical to those in the experimentalgroups.

Magnetic Resonance Imaging

In vivo magnetic resonance imaging was performed at 1.5 Tesla (GEMedical Systems, Milwaukee, Wis.) with an eight-channel phased arraycoil at the specified time points. Scanning was performed with the kneesplaced maximum extension (between 30 and 45 degrees of flexion).Conventional MR included multiplane T1, FSE PD and T2 weighted images.Field of view (FOV): 16-18 cm, matrix: 256×256, (repetition time/echotime) TR/TE: 400/16, 2500/32, 3000/66 msec, echo train length (ETL): 8,bandwidth (BW): 15 kHz, slice thickness: 3, interslice gap: 1 mm).Perfusion was evaluated by using spoiled gradient echo sequence(TR/TE=200/2 ms, flip angle=60, 3 mm slice thickness, and 0.625 mm inplane resolution) with an intravenous contrast agent (Magnevist; Berlex,Wayne, N.J.) 0.2 ml/kg injected 10 s after the start of scan. Fiveimages were obtained per slice, 78 s apart. Post contrast T1-weightedimages were obtained (FOV:16 cm, matrix: 256×256, TR/TE: 400/9 msec,slice thickness: 3 mm, interslice gap: 1 mm) in the coronal and sagittalplanes.

Biomechanical Testing

The bone-ligament-bone ACL complex from both knees for each pig wastested in uniaxial tension. In brief, testing was performed with theknee flexed at 30 degrees of flexion and at room temperature.Immediately after preconditioning, each specimen was tested to failurein uniaxial tension at 20 mm/min. Close-range digital images wereacquired at 3 Hz using a high resolution digital camera with a macrolens (PixeLINK PLA662 Megapixel Firewire camera, PixeLINK, Ottawa ON,Canada) to determine failure mode. The yield load, displacement atyield, tangent modulus (maximum slope of force-displacement curve),maximum load at failure, displacement at failure and total work tofailure (area under force-displacement curve) were determined from theforce-displacement curve measured for each bone-ligament-bone ACLcomplex. The yield load represented the point along the normalizedforce-displacement curve where the mechanical behavior of the ACLcomplex departed from “linear” behavior and for the purposes of thisanalysis was defined as the point where the tangent modulus declines byat least 2% from its maximum value. The displacement at yield was thedisplacement recorded at this same point. The maximum load is themaximal normalized load sustained by the ACL complex prior to failureand the displacement at failure the displacement recorded at the maximumload. The energy to failure was derived by integrating the total areaunder the force-displacement curve.

Statistical Analysis

Mechanical testing measurements were compared at 4 weeks in vivo betweenintact ACL and ACLs treated by suture anchor repair alone and to thosetreated by suture anchor repair plus collagen sponge using F-tests frommultivariate analysis of variance (MANOVA) with 95% confidence intervals(CI). A F-test exceeding the critical value of 3.84 would be regarded asevidence for statistical significance. Each of the six variables (loadat yield, maximum load, displacement at yield, displacement at failure,tangent modulus, and energy to failure) followed a normal(Gaussian-shaped) distribution and therefore data are presented in termsof the mean and standard deviation (SD). Paired t-tests were used toevaluate differences in ACLs treated with suture anchor repair alonecompared to the bilateral side receiving suture anchors with PRP.Statistical analysis was performed using SPSS version 14.0 (SPSS Inc.,Chicago, Ill.). All values of p<0.05 were considered statisticallysignificant.

Results Hematology

A one milliliter sample of whole blood from each pig, and a onemilliliter sample of each of the PRPs were taken to the CBR Institutefor Biomedical Research (Boston, Mass.) and a complete blood count wasperformed. The results are summarized in Table 4.

TABLE 4 Summary of Blood Results Whole Blood Platelet Rich Plasma PRP #1Platelet Count (Platelets/μl) 3.71 × 10⁵ 6.52 × 10⁵ Red Blood Cell Count(RBC/μl) 4.46 × 10⁶   3 × 10⁴ White Blood Cell Count (WBC/μl)  6.6 × 10³ 2.2 × 10³ Hematocrit (%) 28.4 0.2 PRP #2 Platelet Count (Platelets/μl)3.30 × 10⁵ 7.76 × 10⁵ Red Blood Cell Count (RBC/μl) 5.78 × 10⁶   4 × 10⁴White Blood Cell Count (WBC/μl)  9.6 × 10³  2.7 × 10³ Hematocrit (%)39.3 0.3

Mechanical Testing

1.) the Effect of Mixing Time

There was no statistical difference between mixing the components of thegels for 30 seconds or 60 seconds as measured by the maximum elasticmodulus (112±34 Pa vs 142±25 Pa). The results are shown in FIG. 13A.However, mixing for 120 seconds significantly decreased the maximumelastic modulus to only 5±2 Pa (single variable ANOVA p<0.01). Similarfindings were noted for the inelastic modulus (shown in FIG. 13B). Theinelastic modulus for the samples mixed for 30 seconds was 27±8 Pa, andthe inelastic modulus for the samples mixed for 60 seconds was 35±7 Pa.This did not represent a statistically significant difference. However,the inelastic modulus was significantly lower for the 120 second mixingsamples (2±0.4 Pa) when compared to the 30 second and 60 second mixingtime samples (single variable ANOVA p<0.001).

There was no statistically significant difference in the rate ofgelation as measured by time to 45°, the time to G′max, and the time toG″max for the samples mixed for 30 or 60 seconds. For the samples mixedfor 30 seconds, the time to 45° was 3.1±0.0 mins, the time to G′max was16±2.6 mins, and the time to G″max was 16±2.8 mins. For the samplesmixed for 60 seconds, the time to 45° was 2.7±0.4 mins, the time G′maxwas 14.2±4.2 mins, and the time to G″max was 14.2±4.3 mins. However, thesamples mixed for 120 seconds had a time to 45° of 0.3±0.03 mins, whichrepresents a statistically significant decrease (single variable ANOVAp<0.001) when compared to both the 30 second and 60 second mixingsamples. Time to G′max (9.5±5.2 mins) and G″max (8±6.8 mins) were notstatistically significant when comparing the 30 second and 60 secondssamples to the 120 second samples.

2.) the Effect of Mixing Speed

Examining the affect that mixing speed had on the rheological propertiesof the collagen-PRP hydrogels, there was no statistically significantdifference between the three mixing speeds for both the elastic (FIG.14A) and the inelastic modulus (FIG. 14B) (single variable ANOVA).Mixing at 50 RPMs resulted in an elastic modulus of 140±42 Pa, and aninelastic modulus of 35±11 Pa. Mixing at 100 RPMs resulted in an elasticmodulus of 112±41 Pa, and an inelastic modulus of 27±10 Pa. Mixing at200 RPMs resulted in an elastic modulus of 112±30 Pa, and an inelasticmodulus of 27±7 Pa.

Furthermore, the various mixing speeds did not have a statistical affecton the speed at which gelation occurred as measured by time to 45°, timeto G′max, and time to G″max. For the gels mixed at 50 RPMs, the time to45° was 2.3±0.0 mins, the time to G′max 16±2.4 mins, and the time toG″max was 16±2.5 mins. For the gels mixed at 100 RPMs, the time to 45°was 2.8±0.5 mins, the time to G′max 16±1.6 mins, and the time to G″maxwas 16±1.7 mins. For the gels mixed at 200 RPMs, the time to 45° was2.7±0.6 mins, the time to G′max 17±4.1 mins, and the time to G″max was16.7±3.6 mins.

3.) the Effect of Heating Rate

Increasing the heating rate of the collagen-PRP hydrogels did not have astatistical effect on the viscoelastic modulus of the gels. The gelsheated at 9 mV recorded an elastic modulus of 106±20 Pa (FIG. 15A), andan inelastic modulus of 25±4 Pa (FIG. 15B). The gels heated at 11 mV hadan elastic modulus of 93±13 Pa, and an inelastic modulus of 22±3.1 Pa.The gels heated at 13 mV had an elastic modulus of 89±56 Pa, and aninelastic modulus of 22±16 Pa. None of these represent statisticallysignificant differences.

For the gels heated at 9 mV, the time to 45° was 3.6±0.4 mins, the timeto G′max 17.4±1.0 mins, and the time to G″max was 16.4±2.0 mins. Thegels heated at 11 mV had a time to 45° of 2.4±0.6 mins, a time to G′maxof 16±0.5 mins, and a time to G″max of 15±0.8 mins. Finally, the gelsheated at 13 mV had a the time to 45° of 2.0±0.5 mins, a time to G′maxof 17±1.2 mins, and a time to G″max of 16±1.5 mins. Comparing thesevalues, the only statistically significant comparison was between the 9mV and the 13 mV heating rates for time to 45° (single variable ANOVAp<0.01).

4.) the Effect of Injection Temperature

An increase in injection temperature resulted in a decrease in themechanical properties of the Collagen-PRP hydrogels. The gels injectedonto the rheometer plate between 24° C. and 26° C. had an elasticmodulus of 156±26 Pa (FIG. 16A), and an inelastic modulus of 39.4±7.2 Pa(FIG. 16B). The gels injected onto the rheometer plate between 26° C.and 28° C. had an elastic modulus of 128.7±15.7 Pa, and an inelasticmodulus of 32.3±2.7 Pa. The gels injected onto the rheometer platebetween 28° C. and 30° C. had an elastic modulus of 90.3±11.4 Pa, and aninelastic modulus of 22.3±3.6 Pa. Finally, the gels injected onto therheometer plate between 30° C. and 32° C. had an elastic modulus of54.6±30.4 Pa, and an inelastic modulus of 14.2±6.5 Pa. For the elasticmodulus, there was a statistically significant difference between thegels injected between 24° C. and 26° C., and all other groups (singlevariable ANOVA p<0.01), and a statistically significant differencebetween the gels injected between 26° C. and 28° C., and the gelsinjected between 30° C. and 32° C. For the inelastic modulus, there wasa statistically significant difference between the gels injected at 24°C.-26° C., and the gels injected at 28° C.-30° C., and the gels injectedat 30° C.-32° C. (single variable ANOVA p<0.005). There was also astatistically significant difference in inelastic modulus between thegels injected at 26° C.-28° C. and the gels injected at 30° C.-32° C.(single variable ANOVA p<0.005).

The rate of gelation, as measured by time to 45°, the time to G′max, andthe time to G″max was affected by increasing temperature of injection.For the gels injected between 24° C. and 26° C., the time to 45° wasfound to be 2.3±0.1 mins, the time to G′max was 14.6±4.5 mins, and thetime to G″max was 14.5±4.7 mins. For the gels injected between 26° C.and 28° C., the time to 45° was 1.6±0.3 mins, the time to G′max was10.5±3.1 mins, and the time to G″max was 9.4±2.1 mins. For the gelsinjected between 28° C. and 30° C., the time to 45° was found to be1.5±0.0 mins, the time to G′max was 10.6±3.3 mins, and the time to G″maxwas 9.0±2.1 mins. Finally, for the gels injected between 30° C. and 32°C., the time to 45° was found to be 1.0±0.2 mins, the time to G′max was8.6±0.8 mins, and the time to G″max was 8.5±0.9 mins. Statistically,there were no significant differences between the groups for time toG′max and time to G″max; however, there were statistically significantdifferences between the groups for time to 45°. There was a significantdecrease in time to 45° when comparing the gels injected between 24° C.and 26° C. to all the other injection temperature groups (singlevariable ANOVA p<0.003) (FIG. 17). There was also a significantdifference for the time to 45° between the 26° C.-28° C. group and the30° C.-32° C. group, and between the 28° C.-30° C. group and the 30°C.-32° C. group (single variable ANOVA p>0.005).

In vivo results: Effect of injection temperature on repair strength at14 weeks is shown in FIG. 18. Average strength in the sponge alonecontrol group was 206N. The temperature of the gel at injectionsignificantly affects both the in vitro mechanical properties of thehydrogel, as well as the in vivo properties of tissue healing induced bythe hydrogel. Temperatures of less than 26° C. yielded the strongestgels in vitro and temperatures of 28° C. yielded the strongest in vivohealing ligaments.

Example 6 Platelets Enhance ACL Graft Strength and Post Operative KneeLaxicity in a Caprine Model

ACL injuries affect over 200,000 patients each year in the US. While ACLreconstruction is a reliable procedure for grossly restoring stabilityof the knee, normal biomechanics of the knee are not restored and aclinically relevant percentage of patients have excessive laxitypost-operatively. The early healing of the ACL reconstruction graft withdecreased structural properties could explain in part previous work inhumans showing the majority of the increase in knee laxitypost-operatively occurs in the first several months after surgery. Thus,strategies which could improve the early structural properties (strengthand stiffness) of the ACL graft are desirable as a potential solution toreduce the risk of abnormal knee laxity after ACL reconstructivesurgery.

We have demonstrated by histological evaluation of healing of abiomechanically stable partial ACL injury model the growth factorprofile. In normal extraarticular healing of medial collateral ligament(MCL) and patellar tendon (PT) the expression of and timing wasqualitatively similar to placement of a collagen-platelet hydrogel inthe partial ACL. This is in contrast to the lack of healing and severelylimited growth factor expression in the partial ACL injury without(control) collagen-platelet hydrogel. Specific individual growth factors(PDGF vs TGF and EGF vs VEGF) have been applied to ACL reconstructionmodels in either sheep or canines. Both growth factors that are abundantin platelets (PDGF and TGF) demonstrated improved load and stiffness at12 weeks postoperatively. However the application of a growth factor tostimulate revascularization (VEGF) weakened the graft at 12 weeks. Theearlier time points (6 weeks), optimal dosage, combination orapplication method to improve early structural properties when the ACLreconstruction graft is near the weakest is unknown. Thus, the strategyto apply the body's own growth factors to promote the return ofstructural properties of the ACL reconstruction graft requires furtherresearch.

This study was performed to show that placement of a platelet gel aroundan ACL graft at the time of surgery would improve the early mechanicalproperties of the graft (maximum load and stiffness). It was also shownthat the platelet concentration around the graft would have a directcorrelation with the early load to failure of the ACL graft.

Materials and Methods: Animal Model

Twelve 4-year old castrated male Nubian cross goats underwent unilateralanterior cruciate reconstruction using a bone-patellar tendon-boneautograft. In the experimental group six goats had the graft augmentedwith a collagen-platelet hydrogel, while the six control goats hadaugmentation with the collagen-hydrogel only. The surgeries werealternatively performed between the right and left knees within eachtreatment group. The animals were allowed unrestricted cage activitywhile ACL reconstruction grafts were healing. At six weekspostoperatively they were euthanized with an overdose of pentabarbitolsolution (Euthasol; 1 cc/10 lbs). At the time of euthanasia, both thereconstructed and contralateral control knees were harvested and storedat −20° C. prior to mechanical testing.

Surgical Procedure

The animals were tranquilized preoperatively using acepromizine (10 mgIM). Anesthesia was then induced with sodium pentothal (5-8 mg/Kg IV)and maintained during surgery using isofluorane.

A 15 blade was used to make an incision from top of patella to below thetibial tubercle just medial of midline. The prepatellar bursa was cut inline with the skin incision to expose the paratenon. A longitudinal cutwas made centrally in the paratenon to expose the patellar tendon. Themedial and lateral borders of the patellar tendon were palpated and a 6mm wide graft marked with the electrocautery. The patellar block was15×6 mm, leaving 10 mm of patella intact superiorly. The tibial boneblock was 10×6 mm. The harvested graft was shaped to fit 6 mm diameterbone blocks and 1.5 mm drill holes were placed in the bone block on eachside. Because the length of the patellar tendon is greater than the ACL,the tibial bone block was folded over onto the patellar tendon andsutured in place to make an 8 mm bone-tendon block on this side toshorten it. Two #2 Ethibond sutures were placed in each bone block. Theintracondylar notch was exposed through the central defect in thepatellar tendon by sectioning the fat pad. The intermeniscal ligamentwas not cut. A Lachman test was checked for baseline stability of theknee. A #11 blade was used to release the ACL from the back of thenotch, and the ACL was removed by releasing the ligament from its tibialinsertion. A manual Lachman was performed to verify complete functionalloss of the ACL. The tibial tunnel was drilled using the tibial aimingguide set at 65°. The pin was over-drilled with an 8 mm drill and allsoft tissue removed. A notchplasty was performed using a curette. Theknee was hyperflexed and a 6 mm offset femoral drill guide (Arthrex Inc,Naples Fla.) was placed into the back of the notch at the 10:30position. The passing pin was drilled through the femur and thenover-drilled with a 7 mm drill to 20 to 25 mm. Integrity of the backwall of the femoral tunnel was verified in all cases. The graft wasplaced into the femoral tunnel first using the Ethibond sutures, andthen secured in the femur using a 5×20 mm interference screw (Arthex,Inc.). The graft was then pulled retrograde into the tibial tunnel. Withthe knee at 60 degrees of flexion, the graft was firmly tensioned andsecured in the tibial tunnel using a 6×20 mm interference screw (Arthex,Inc.). Tibial fixation was augmented with sutures to the periosteum ifthe tibial fixation was not deemed stable enough.

For the experimental group the graft was augmented with a collagensponge placed between the ACL and LFC using a freer elevator, with partof the sponge lying anteriorly to the graft. Two cubic centimeters of acollagen-platelet hydrogel (n=6) was placed over the sponge. The controlgroup was identical except no platelets were added to thecollagen-hydrogel. After ten minutes, the knee was closed in layers. Theanimals were kept under anesthesia for 1 hour after gel placement tomaintain the knees in the resting position and allow complete gelation.

Post-operative analgesia was control using Buprenorphine (0.01 mg/Kg IM,twice daily) and Ketoprofen (1 mg/Kg IM, once daily) for five days.Ampicillin (10 mg/Kg SC, twice daily) was administered for 10 days toreduce the risk of infection.

Collagen Gel and Collagen-Platelet Gel Manufacture

Rat tail collagen was acid-solubilized as described herein. For thecollagen group, the collagen was neutralized to a pH of 7.4 and added tothe surgical site just after neutralization. To add platelets to thegel, initially the production of platelet-rich plasma was attempted;however, due to the similarity in size and weight of the caprineplatelet and red blood cell, centrifugation protocols using 150 to 250 gbetween 20 and 30 minutes all resulted in effective decrease of redblood cells in the PRP, but platelet counts in the PRP fraction wereless than 100% that of the whole blood with all protocols. Using themost effective protocol determined ex vivo, 250 g for 30 minutes, theplatelet yield in the caprine PRP averaged 102%+/−68% (mean+/−standarddeviation) for the 12 goats in this study when the measured MPV was usedto calculate platelet number. In addition, there were large variationsin enrichment seen within the group. Therefore, we elected to use wholecaprine autologous blood for the collagen-platelet group. This resultedin the peripheral blood platelet concentration determining theconcentration of platelets delivered in the collagen-platelet hydrogel.In addition, we measured the platelet concentration in the peripheralblood for all animals, both experimental and control groups. Fifty-fourcc of blood was drawn from each animal into a syringe containing 6 cc ofacid-citrate-dextrose as an anticoagulant. At the time of gel placement,the collagen was neutralized and mixed with the blood in a 4:1collagen:blood ratio and the collagen-platelet gel added to the graft.

Mechanical Testing

After all specimens were collected, the knees were thawed and preparedfor laxity and failure testing. The soft tissues surrounding the tibiaand femur were dissected free leaving the joint capsule intact. Thedistal tibia and proximal femur were then potted in PVC pipes using apotting material (SmoothCast 200; Smooth-On, Easton Pa.) so that theycould be mounted for mechanical testing.

The anteroposterior load-displacement responses of the intact jointswere measured using a custom designed fixture with the knee locked at30° and 60° of flexion (FIG. 19) (Fleming et al JOR 19: 841, 2001).Anterior and posterior directed shear loads of ±60 Newtons were appliedto the femur with respect to the tibia using a MTS 810 Materials TestingSystem (MTS, Prairie Eden, Minn.) while the AP displacement wasmeasured. Axial rotation of the tibia was locked in the neutralposition, and all other motions were left unconstrained.

After completing the AP laxity tests, the tibia and femur werepositioned on the so that the mechanical axis of the ACL was collinearwith the load axis of the material test system (Woo et al; AJSM 19: 217,1991; Tohyama et al; AJSM 24: 608, 1996). The knee flexion angle was setat 30°. The tibia was mounted to the base of the MTS via a sliding X-Yplatform. The femur was unconstrained to rotation. This enabled thespecimen to seek its own position so that the load was distributed overthe cross section of the healing graft when the tensile load wasapplied.

The joint capsule, menisci, collateral ligaments and the PCL weredissected from the joint leaving the ACL graft and scar mass intact. Thefemur-graft-tibia complexes were then loaded in tension to failure at 20mm/min while the failure load-displacement data were recorded. Identicalprotocols were performed on the contralateral ACL-intact knees. From theMTS load-displacement tracing, the failure load, failure displacement,and the linear stiffness were determined.

Exclusion

The first animal operated on was assigned to the collagen-plateletgroup. There were technical difficulties with the graft harvest in thisanimal and at the conclusion of surgery, it was decided to exclude thisanimal from the analysis. In addition, one of the animals in thecollagen alone group had a graft with a failure strength more than threetimes higher than any other animal in either group, thus was excludedfrom the remaining analysis due to the 3 sigma rule. Therefore, neitheranimal is represented in any group statistic nor are they graphicallydepicted in any figure.

Statistical Analyses

Comparisons of AP laxity values, failure strength, and stiffness valuesbetween the collagen (carrier only) and platelet collagen groups weremade. The differences for each of these parameters between the treatedknee and the contralateral control knee were calculated. Unpairedt-tests were performed to determine if the differences were significant(p<0.05). Correlation analyses were performed to determine theassociation between systemic platelet count and the strength of thegraft after 6 weeks of healing.

Results Surgical Outcome

Eleven out of the 12 animals recovered well from surgery. However, onegoat from the collagen only group died within one day of surgery.Autopsy revealed extensive atherosclerosis and the cause of death wasthought to be cardiac-related. At the time of euthanasia, all animalsappeared to be walking normally.

Gross Appearance

Observers were blinded to group when harvesting and grading thespecimens. There was no difference between the collagen andcollagen-platelet groups on gross appearance in terms of rate ofreformation of the ligamentum mucosum, rate of scar or adhesionformation from the notch scar mass to the harvest defect of the patellartendon or amount of joint adhesions observed. There was no differencebetween the groups in whether the scar mass infiltrated only the mostcranial section of the graft or whether it bridged from femur totibia—both findings were seen in ligaments of both groups.

Biomechanics

At a knee flexion angle of 60 degrees of flexion, the AP laxity of theknees was 34% lower in the collagen-platelet group than in the collagengroup, a difference which was statistically significant (17.2+/−3.3 mmvs 23.1+/−4.0 mm; mean+/−SD; p<0.05). At 30 degrees of flexion, therewas also a 40% decrease in AP laxity of the knees in thecollagen-platelet group; however, the difference approached, but did notreach, statistical significance, due in part due to the large standarddeviations seen within each group (collagen-platelet group 14.3+/−4.0 mmvs collagen group 20+/−4.5 mm; p<0.09).

The collagen-platelet group strength was 30% higher than the collagengroup (139+/−41N vs 108+/−47N; both mean+/−SD), a difference that wasnot statistically significant (p>0.30). The values in both groups wereapproximately 10% of the intact ACL strength. Femoral tunnel size,collagen sponge size and collagen gel amount were not found to besignificant predictors of failure strength.

There was no significant difference between the groups in terms offailure displacement. The collagen group failed at 9.3+/−5.1 mm(mean+/−SD) and the collagen-platelet group failed at 8.4+/−4.4 mm(p>0.80). Interestingly, both values were far lower than the failuredisplacement in the contralateral intact ligaments, which averaged20.8+/−1.8 mm. There was also no significant difference in linearstiffness between groups, with the collagen group having a stiffness of22+/−15 N/mm and the collagen-platelet group having a stiffness of26+/−14 N/mm (mean+/−SD; p>0.60). Both groups were less than one thirdof the intact ligament average stiffness (90+/−34 N/mm; mean+/−SD).

Higher systemic platelet counts correlated significantly with bothhigher failure load (FIG. 20: R̂2>0.67) and higher ligament linearstiffness (FIG. 21A and FIG. 21B: R̂2>0.80) using a linear regressionmodel. There was no significant correlation between plateletconcentration and failure displacement (R̂2=0.40) or AP laxity at 60degrees (R̂2=0.44) with the number of animals tested. The data isdepicted graphically in FIG. 22 and FIG. 23A and FIG. 23B. FIG. 22 is agraph depicting strength of the joint as a function of platelet count.FIGS. 23A and 23B are bivariate scattergrams wit regression 95%confidence bands. FIG. 23A depicts fail load as a function of plateletcount. FIG. 23B depicts stiffness as a function of platelet count.

Thus, AP laxity measured at 30 degrees of knee flexion was significantlyimproved in the experimental platelet group when compared to thattreated with the carrier alone (154%+/−44% vs 355%+/−55%; p=0.03). Therewas no significant difference between the maximum load of the ACL graftin the two groups; however, the maximum load of the graft correlateddirectly with the systemic platelet count in both the experimental(R2=0.95) and control (R2=0.85) groups. The addition of platelet to thecollagen-hydrogel improved AP laxity when compared to previous reportsof ACL reconstructed knees at six weeks.

The addition of blood platelets to the collagen hydrogel (experimentalgroup) resulted in clinically significant reduction in knee laxity atsix weeks after autograft patellar tendon ACL reconstruction. Inaddition, the systemic platelet count of the animals correlated directlywith the maximum load for both the experimental group (collagen-platelethydrogel) and the control group (collagen hydrogel). Both of thesefindings highlight the role blood platelets play in ACL reconstructiongraft healing with presumably clinically relevant reduction in earlyundesired postoperative AP laxity.

The improvement in AP laxity was evident when the knees were tested in30 degrees of flexion (full extension in the caprine knee). At thisposition, the knees treated with collagen-platelet hydrogel had only 54%more AP laxity than the contralateral knees with intact ACLs, while theknees treated with collagen hydrogel alone were over 200% more lax thanthe intact knees. At 60 degrees of flexion, the difference betweengroups was smaller and insignificant. This finding suggests a potentialrole for reducing early undesired clinical laxity. Whether theplatelets' effect was on the graft healing or capsular structures isunknown. Further optimization of platelet concentration as well astiming of administration is needed. The large increases in AP laxityseen in the collagen hydrogel alone group are consistent with those ofprevious studies of ACL reconstruction with autogenous patellar tendongrafts using the goat model (Abramowitch JOR 21:707, 2003; Cummings JOR20:1003, 2002; Papageorgiou AJSM 29:620, 2001; Jackson AJSM 21:176,1993). Papageorgiou et al reported 238% and 285% increases in AP laxitywith the knee at 30 degrees and 60 degrees of flexion, respectively,after 6 weeks of healing. The improvement seen in the collagen-plateletgroup is also a marked improvement from results published previously onACLR in sheep where the AP laxity of the knee increased from 2.0+/−0.7mm in the intact knee, to 8.3+/−2.3 mm at six weeks in a model usingfemoral and tibial interference screw fixation as we did in this study(HUNT et al Knee Surg Sports Traumatol Arthrosc. 2006 December;14(12):1245-51.).

The six week time point is well recognized as a nadir of strength in ACLreconstruction for animal models. The strength values in both groupswere approximately 10% of the intact ACL, which is slightly higher thanprevious reports of 3% in the sheep model (Hunt et al Knee Surg SportsTraumatol Arthrosc. 2006 December; 14(12):1245-51.), and typical for thegoat model after 6-weeks of healing (Papgeorgiou AJSM 29:620, 2001;Abramowitch 21:708, 2003). By applying platelets in a stabilizedcollagen-hydrogel we have simulated the “natural” environment found withplatelets being deposited in a fibrin clot for extraarticular healing.Further, platelets contain a multitude of growth factors in addition toTGF-β and PDGF in the appropriate concentration for extraarticularhealing. Additionally, the cost of recombinant TGF or PDGF or EGF farexceeds the cost to apply autograft platelets from blood.

Limitations of the study include the inability to control rehabilitationin the animals and the inability in the caprine model to provide two- tofour-fold increased platelet concentrates in the collagen hydrogel.Ruminants must be upright standing in the very early post-operativeperiod, thus it is difficult in this model to protect the ACL graft fromweight bearing loads. Bandaging and immobilization are not practical oreffective in this animal model. The inability to concentrate the bloodplatelets limits the ability to discover whether increasing plateletcount above that of whole blood would continue to enhance ACL grafthealing and reduce postoperative laxity further. However, regardless ofthe limitations of the model, the data clearly demonstrates that theaddition of platelets enhances graft healing and improves AP stabilityof the knee after ACL reconstruction at an early time point in thecaprine model.

Example 7 Suture Techniques that Restore Normal AP Laxity of the Kneeafter ACL Transection

The data described herein demonstrate suture techniques that go fromfemur to tibia can restore the normal AP laxity of the knee at timezero, particularly if they are tied in a small amount of flexion and thetibial attachment point is within the normal ACL footprint. As shownbelow, repair to the tibial stump of the ACL (Marshall technique)resulted in knees with over 5 mm greater AP laxity than knees with anintact ACL. Suture repair to bone using fixation points within thenormal ACL footprint resulted in knee laxity within 0.5 mm of the kneeswith an intact ACL when the sutures were tied with the knee flexed at 60degrees. Laxity increases of 1 to 3 mm were seen if the sutures weretied with the knee in 30 degrees of flexion or in more posteriortunnels.

Primary repair of the ACL was pioneered by John Marshall in the 1960's(Marshall J L, Warren R F, Wickiewicz T L, Clin Orthop. 1979;143:97-106, Marshall J L, Warren R F, Wickiewicz T L. Am J Sports Med.1982; 10:103-107.). Favor for this technique was lost due to the highre-rupture rate (Feagin J A, Jr., Curl W W. Am J Sports Med. 1976;4(3):95-100.) and the limited improvement over nonsurgical treatmentseen in patients undergoing primary repair (Engebretsen L, Benum P,Fasting O, Molster A, Strand T. American Journal of Sports Medicine.1990; 18(6):585-590, Grontvedt T, Engebretsen L, Benum P, Fasting O,Molster A, Strand T. Journal of Bone & Joint Surgery—American Volume.1996; 78(2):159-168.). However, recent discoveries by the inventor havesuggested that amplifying the repair response of the torn ACL using abioengineered scaffold to deliver growth factors into the wound site mayresult in functional healing of the severed ACL (Murray M M, Spindler KP, Devin C, et al. J Orthop Res. April 2006; 24(4):820-830, Murray M M,Spindler K P, Ballard P, Welch T P, Zurakowski D, Nanney L B. J OrthopRes. Apr. 5, 2007).

Since Marshall's work outlining his technique of primary repair, therehas not been much interest or published work on comparison of suturetechniques for primary repair of the ACL. This is in stark contrast tothe numbers of papers published each year on ACL reconstructiontechnique, including papers on fixation types, tunnel placement andnumber of bundles to reconstruct. With the recent renewed interest inprimary repair, additional work is needed to define the AP laxity of theknee after suture repair of the ACL using various techniques. Definingthese surgical variables will allow for more accurate testing of newtissue engineered constructs in animal models, and also begin to definethe most appropriate techniques for eventual human use.

In this study two hypotheses were tested. A first hypothesis was thatsuture repair could restore the normal AP laxity of the knee at timezero, and a second hypothesis was that the angle of flexion at which thesutures were tied would have a significant effect on the resultant APlaxity of the knee.

Materials and Methods

Six hindlimbs were retrieved from 30 kg female Yorkshire pigs at thetime of euthanasia for other IACUC approved studies. The limbs werefrozen until the time of testing (approximately 3 weeks). The limbs werethawed in warm water on the morning of testing. The knees were isolatedby sectioning the femur just below the lesser trochanter and sectioningthe tibia 2 cm above the ankle joint. The muscular attachments to tibiaand femur were removed with care taken not to violate the knee jointcapsule. All extra-capsular muscle was also removed. The femur and tibiahad drywall screws placed at intervals along the bone (four screws ineach bone) to assist with purchase in the potting material, then thebones were sequentially potted in 2″ diameter polyvinyl chloride pipingusing Smooth-On casting material. Knees were wrapped in towels moistenedwith normal saline until testing.

The AP laxity testing was performed using a customized jig mounted on anInstron testing machine (FIG. 24). The femur was mounted in a movablefixture, which allowed for positioning the knee in 60 degrees offlexion. The pig knee extends only to 30 degrees short of fullextension, thus the 60 degree position was thought to correspond to the30 degree position in humans. A pilot study demonstrated that the 30degree position was less sensitive to changes in AP laxity in the kneeand therefore, only the 60 degree position was used in this study. Oncethe knee was positioned in the fixture, a cyclic load of 30N was appliedto the femur. This resulted in an anterior femoral displacement at 30Nfollowed by a posterior femoral displacement relative to the tibia to30N. The magnitude of the displacements as load was applied was measuredfor each cycle at 100 Hz and plotted using Excel to give aload-displacement curve (FIG. 25).

Each of the six knees was tested in the intact state (INTACT). Afterthat testing, dissection was performed to remove the patella andpatellar tendon and expose the notch and the testing repeated (PATDEFICIENT). The specimen was brought back to the dissection table andthe ACL completely transected and testing repeated (ACL DEFICIENT).

The knees were then prepared for various primary repair techniques.First, a TwinFix 3.5 mm titanium anchor (Smith-Nephew, etc) was placedin the posterolateral notch of the femur, at the 11:00 position for theright knees and the 1:00 position for the left knees. This anchor hadtwo Durabraid sutures passed through the anchor eyelet, resulting infour strands available for repair. These sutures were used for alltests. A four-stranded Marshall repair technique was performed bypassing two looped #1 Vicryl sutures through the tibial stump at variousdepths and securing these four ends to the four ends of the Durabraidfor a four-stranded Marshall repair. The sutures were first tied withthe knee first in 30 degrees of flexion (MARSHALL 30) and then in 60degrees of flexion (MARSHALL 60). The sutures were unknotted and thetibial stump of the ACL resected to reveal the tibial insertion sites.In the pig, there are two discrete tibial insertion sites of the ACL—oneis posterolateral, behind the anterior horn attachment of the medialmeniscus, and the second is anteromedial, located between the anteriorhorn attachment of the medial meniscus and the anterior horn attachmentof the lateral meniscus (FIG. 26). A tibial aimer (ACUFEX) was used toplace a 2.4 mm guide pin from the anteromedial border of the tibia up toeach of these insertion sites. Care was taken to maintain a minimum of 5mm between each of the drill sites on the anteromedial tibia. A thirddrill hole was made just medial to the apex of the lateral tibial spine.These tibial drill sites were labeled ANTERIOR for the insertion of theAM bundle, MIDDLE for the insertion of the PL bundle and POSTERIOR forthe lateral tibial spine site. In four of the knees, drilling actuallywent through the potting material to get to the appropriate site.

AP laxity testing was then performed with sutures passed through theanterior, middle or posterior bone holes and tied over an endobutton.Sutures through each tunnel were tied in first 30 degrees of flexion andthen 60 degrees of flexion and then underwent AP laxity testing at the60 degree position. For example, each knee had all four sutures placedthrough the anterior tunnel and tied together over an endobutton withthe knee at 30 degrees of flexion. This test was labeled (ANTERIOR 30).The sutures were then untied and re-tied with the knee in 60 degrees offlexion and the AP laxity of the knee measured (ANTERIOR 60). Sutureswere then untied, passed through the middle tunnel, tied at 30 degreesand tested (MIDDLE 30), and so on. In addition to all four sutures goingthrough the anterior, middle and posterior tunnels, a final positionwith two sutures going through the anterior tunnel and two sutures goingthrough the middle tunnel was also tested (ANT-MID 30 and ANT-MID 60).

After each test, the knee was inspected to make sure the suture anchorwas still secure and this was verified. There was no evidence of sutureanchor pullout for any of the tests.

Statistical Analysis

Mixed model ANOVA with suture location and knee flexion angle asrepeated measures terms and subject factors included to track individualspecimens was used to determine the significance of differences betweengroups. A compound symmetry covariance structure (which demonstratedgood fit based on Akaike's Information Criterion (AIC)) was selected.

Results

The intact knees had an AP laxity of 4.9 mm+/−0.4 mm (mean+/−SEM).Removal of the patella, patellar tendon, ligamentum mucosum and fat padhad a negligible effect on the AP laxity of the knee, with values forthat group of 5.2+/−0.3 mm and t-testing p>0.79 for comparison betweenthe two groups). When the ACL was sectioned, the laxity of the kneeexceeded the maximum level set by the testing device (32 mm), and evenat those displacements, no load was placed on the load cell. Therefore,this group was assigned a displacement of 32 mm.

Primary repair using the Marshall technique resulted in improved laxityin comparison with the ACL deficient knee, but increased AP laxity whencompared to the intact and patellar deficient knee when the repair wasdone at both 30 degrees of flexion and at 60 degrees of flexion (Table5). These differences between intact and Marshall technique ligamentknee laxity were statistically significant at both 30 and 60 degrees(p<0.002 for both comparisons).

The AP laxity of the knee after suture repair was dependent on thelocation of the tibial suture (F=35; p<0.001). Sutures placed in themiddle bone tunnel, located within the ACL tibial insertion site,restored AP laxity of the knee to values similar to that in the intactACL knees with the patella removed (5.2+/−0.6 mm vs 5.2+/−0.4 mm,p>0.99; mean+/−SEM). Sutures placed in the anterior bone tunnel resultedin repairs with an average of 1.2 mm greater laxity than sutures placedin the middle location (6.4+/−0.4 mm), a difference which approached,but did not reach statistical significance in this multiple comparisonmodel (p>0.05). Placement of the suture in a more posterior location onthe tibial spine or in the ACL tibial remnant resulted in knees withsignificantly greater laxity than the knees with an intact ACL or kneesrepaired to anterior or middle tunnels (p<0.05 for all comparisons).

FIGS. 27A-27C are photographs of the anterior (27A), middle (27B), andposterior (27C) tibial tunnel positions. FIG. 28 is a graph depicting APlaxity values for all specimens. FIG. 29 is a graph depictingdifferences from intact AP laxity values.

The AP laxity of the knee after suture repair was also dependent on theknee flexion angle when the sutures were tied (F=30, p<0.001). Laxitywas greatest when the repairs were tied at 30 degrees of flexion, withless laxity noted when the repairs were tied at 60 degrees (p<0.001);however, the laxity in both groups remained higher than that of the ACLintact knees (p<0.02).

There was no interaction between tibial suture location and knee flexionangle (p=0.67).

Suture techniques that go from femur to tibia can restore the normal APlaxity of the knee at time zero, particularly if they are tied in asmall amount of flexion and the tibial attachment point is within thenormal ACL footprint. The data demonstrate that suture repair to thetibial stump, as in the Marshall technique, does not restore normal APlaxity of the knee, a finding which may be one of the reasons thistechnique of ACL repair resulted in a large percentage of patientshaving abnormal knee laxity post-operatively.

However, in several of the groups, there were some knees that had lessAP laxity after suture repair than in the intact ACL condition. Thiscould potentially result in overconstraining of the knee. Whetheroverconstraint or excess laxity are more likely to proceed to earlydegenerative joint changes is as yet unclear, but it is likely thatrepairs that result in large changes in knee laxity may not be ideal.

TABLE 5 AP laxity as a Function of Repair Type Std. 95% Confidence MeanError df Interval Lower Upper Lower Upper Lower Location Bound BoundBound Bound Bound Intact 4.933(a) 0.624 59.855 3.686 6.181 Patellar5.200(a) 0.624 59.855 3.952 6.448 Deficient Marshall 12.433(a)† 0.44452.094 11.542 13.325 Anterior 6.400(a) 0.444 52.094 5.509 7.291 Middle5.192(a) 0.444 52.094 4.3 6.083 Posterior 7.658(a)‡ 0.444 52.094 6.7678.55 Ant-Mid 5.808(a) 0.444 52.094 4.917 6.7 (a)Based on modifiedpopulation marginal mean. †Statistically different from the ACL intactknees (p < 0.002). ‡Statistically different from ACL intact knees (p <0.05).

Example 8 Use of 40 Mend High Density Sponges (HDBC Sponges)

A study comparing the effectiveness of standard density collagen sponges(Gelfoam) and high density collagen sponges (HDBC) was performed. 1 cmdiameter sponges of both Gelfoam and HDBC (3× increase in collagenconcentration) were used as the scaffold.

The HDBC sponge was prepared by lyophilizing a collagen slurry andreconstituting it to a density of 40 mg/ml collagen. The slurry wasneutralized and allowed to gel at 38° C. The resulting gels were thenlyophilized. Both GELFOAM and HDBC sponges were seeded with cellssuspended in platelet-rich plasma (PR or cells suspended in collagenslurry +PRP (concentration 1×10⁶ cells/ml for both groups). Both cellsolutions were allowed to absorb into the sponges for 30 min in theincubator, and then 2 drops of complete media was placed on top of thesponges to keep them moist overnight. After 12 hours, 1 ml of completemedia was added to each well.

Results

The standard density sponges (GELFOAM) did not absorb the PRP orcollagen slurry very efficiently. Cell counts within the sponges weremeasured at Day 2 and Day 10 (FIG. 30). The greatest cell proliferationoccurred in the HDBC +PRP group and the least proliferation in theGelfoam +PRP group.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety.

1-19. (canceled)
 20. A composition comprising a sterile solution ofsolubilized collagen in a concentration of greater than 5 and less thanor equal to 50 mg/ml and having a viscosity of 1,000-200,000 centipoise,hydroxyproline in a concentration of 0.1-5.0 μg/ml, a neutralizing agentwherein the composition has an osmolarity of 280-350 mOs/kg, wherein thecomposition is free of thrombin, and any added crosslinking agent. 21.The composition of claim 20, wherein the solution is a liquid or gel.22. The composition of claim 20, wherein the solubilized collagen ispresent in a concentration of greater than 15 mg/ml and less than orequal to 40 mg/ml.
 23. The composition of claim 20, further comprising aplatelet, white blood cells, an antibiotic, an anti-plasmin agent,insoluble collagen, a plasminogen activator inhibitor, aglycosaminoglycan, decorin or biglycan.
 24. The composition of claim 20,further comprising a buffer, wherein the composition has a pH of 6.8-8.0or a pH of 7.4.
 25. The composition of claim 20, wherein the collagen isenzyme solubilized collagen or atelocollagen.
 26. The composition ofclaim 20, wherein the solution is maintained at a temperature of 4° C.27. The composition of claim 20, wherein the composition is prepared bysubjecting a sterile solution of solubilized collagen in a concentrationof greater than 5 and less than or equal to 50 mg/ml and having aviscosity of 1,000-200,000 centipoises to a temperature of at least 30°C. wherein the sterile solution of solubilized collagen forms a collagenscaffold.
 28. The composition of claim 20, wherein the composition formsa collagen scaffold.
 29. The composition of claim 20, wherein thesterile solution of solubilized collagen is mixed for 30-120 seconds orfor 30-60 seconds.
 30. The composition of claim 20, wherein the sterilesolution of solubilized collagen is subjected to a temperature of 24-28°C. and then subsequently is exposed to a temperature of greater than 30°C.
 31. The composition of claim 1, wherein the composition is used fortreating ruptured articular tissue by contacting the ends of a rupturedarticular tissue in a subject with the composition of claim 20, andallowing the solution to set to treat the ruptured articular tissue. 32.A quick set composition comprising a sterile solution of solubilizedcollagen in a concentration of greater than 5 and less than or equal to50 mg/ml and having a viscosity of 1,000-200,000 centipoises and a pH of6.8-8.0, wherein the solution has an osmolarity of 280-350 mOs/kg,wherein the solution sets into a scaffold within 10 minutes of exposureto temperatures of greater than 30° C.
 33. A compressible expandablescaffold, comprising a collagen sponge, wherein the collagen is type Isoluble collagen and wherein the sponge further comprises thecomposition of claim 20.